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Humanity Will Continue to Live an Inferior Life Than What is Possible Until the Two Halves: All Individuals in Them: That Make It are Absolutely Fundamentally and Jubilantly Equal at Liberty
 

 

Year Gamma: London: Thursday: January 04: 2018
First Published: September 24: 2015

Change: Either Happens or Is Made: When It is Not Made It Happens Regardless in Which We Become Mere Logs and Get Washed Away in and by Utterly Mechanical Forces of Dehumanisation: When Made Change is Created by Our Conscious Choices, Efforts, Initiatives and Works: In the Former We Let Go Off Our Humanity So That Dehumanisation Determines and Dictates the Existence of Our Sheer Physiologies: But in the Later We Claim, Mark and Create Our Humanity as to the Change We Choose to Make and Create It Onto Reality: To Nurture, Foster, Support, Sustain, Maintain, Enhance, Expand, Empower and Enrich the Very Humanity That We Are:  As Individuals, As Families, As Communities and As Societies All of Which Now Exist in the Fabrics of Time-Space of What is Called Civic Society: One That Exists by Natural Justice and Functions by the Rule of Law: Ensuring Liberty and Equality, Along with Purpose and Meaning of Existence, Exist in Each and Every Soul Equally at All Times: The Humanion

 

 

 

 

 

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Molecular Biology

Stories Published in Molecular Biology Section in September-December 2015

Thermotaxis: Some Like It Hot, but Not too Hot

In new research published in eLife yesterday, Chloe Greppi, Gonzalo Budelli, Paul A Garrity ( Brandeis University, United States) writes that a temperature-sensitive receptor prevents mosquitoes from being attracted to targets that are hotter than a potential host.

From warm summer days to cold winter nights, temperature is a ubiquitous sensory stimulus. All animals rely on their ability to detect environmental temperatures to avoid harm and to seek out more optimal conditions. Some animals, such as mosquitoes, also use their temperature sensors for a more nefarious purpose: to locate warm prey for a blood meal.

The ability of the mosquito to home in on warm bodies was first recognized over a century ago (Brown, 1951; Howlett, 1910), but the details of this behavior are still not fully understood. Now, in eLife, Roman Corfas and Leslie Vosshall from Rockefeller University report on the molecular basis of temperature-sensing behavior in Aedes aegypti, the mosquito that spreads yellow fever (Corfas and Vosshall, 2015). They show how avoiding high temperatures can stop these insects from being attracted to targets that are too hot to represent a suitable host: in other words, while these mosquitoes like it hot, they don’t like it too hot.

Corfas and Vosshall’s study is grounded in previous work on the fruit fly Drosophila melanogaster (Barbagallo and Garrity, 2015). Fruit flies use at least two molecular receptors to guide their movements in response to warmth (Hamada et al., 2008; Ni et al., 2013), and while about 250 million years of evolution separate flies and mosquitoes, versions of each receptor are present in A. aegypti. It is unclear precisely where these receptors, TRPA1 and GR19, are expressed in Aedes mosquitoes, but in the malaria-spreading mosquito Anopheles gambiae, TRPA1 is expressed at the tips of the antennae (Wang et al., 2009). This is intriguing, because the tip of a mosquito antenna houses very sensitive thermoreceptors that could help drive host-seeking behavior (Davis and Sokolove, 1975).

Female mosquitoes normally prefer temperatures around 23°C. However, a puff of carbon dioxide (which could indicate that a metabolically active host is nearby) drives the mosquitoes to seek out temperatures that are closer to the body temperature of a mammal or bird (that is, between about 37°C and 43°C; McMeniman et al., 2014). Corfas and Vosshall started by further characterizing this heat-seeking behavior. They found that mosquitoes were strongly attracted to a target when it was heated to temperatures above ambient, but only up to ~50°C. When it got hotter, this attraction declined strongly.

To probe the molecular mechanisms that might control this response, Corfas and Vosshall exploited genome-editing techniques to knock out the genes for GR19 and TRPA1 in A. aegypti. They found that mosquitoes lacking GR19 behaved like wild type mosquitoes and showed normal responses to heat. However, mosquitoes without TRPA1 continued to be attracted to the target even when its temperature reached potentially harmful levels (> 50°C).

While the ability of animals to avoid high temperatures is commonly viewed from the perspective of damage avoidance, Corfas and Voshall raise the possibility that this response could also help a heat-seeking mosquito to choose among multiple potential targets. In fact, when tested for their ability to discriminate between two hot targets (one at 40°C, the other at 50°C), TRPA1-knockout mosquitoes showed a greatly reduced preference for a 40°C target over a 50°C target. Thus, while TRPA1 is not required for heat seeking, per se, it helps to set an upper limit on the temperatures mosquitoes seek, which prevents them from being attracted to stimuli warmer than their hosts.

While the work of Corfas and Voshall advances our understanding of temperature sensing in A. aegypti, the identity of the receptor (or receptors) that drives heat-seeking behavior remains elusive. However, the recent use of rapid genome-editing techniques in Aedes mosquitoes (including the CRISPR-Cas9 system; Kistler et al., 2015), has dramatically simplified the study of the function of genes in this organism. This greatly increases the likelihood that more of the molecules behind heat seeking and other mosquito sensory responses will be uncovered in the near future. As mosquito-borne illnesses kill more than a million people every year, interventions that can reduce the spread of such diseases are crucial. It is hoped that an increased understanding of how mosquitoes target their hosts can help accelerate the development of new control strategies.

The Article can be read in eLife   DOI

The Cation Channel TRPA1 Tunes Mosquito Thermotaxis to Host Temperatures



In new research published in eLife Román A Corfas, Leslie B Vosshall ( The Rockefeller University, United States; Howard Hughes Medical Institute, The Rockefeller University, United States) writes that while most animals thermotax only to regulate their temperature, female mosquitoes are attracted to human body heat during pursuit of a blood meal. Here we elucidate the basic rules of Aedes aegypti thermotaxis and test the function of candidate thermoreceptors in this important behavior. We show that host-seeking mosquitoes are maximally attracted to thermal stimuli approximating host body temperatures, seeking relative warmth while avoiding both relative cool and stimuli exceeding host body temperature. We found that the cation channel TRPA1, in addition to playing a conserved role in thermoregulation and chemosensation, is required for this specialized host-selective thermotaxis in mosquitoes. During host-seeking, AaegTRPA1-/- mutants failed to avoid stimuli exceeding host temperature, and were unable to discriminate between host-temperature and high-temperature stimuli. TRPA1-dependent tuning of thermotaxis is likely critical for mosquitoes host-seeking in a complex thermal environment in which humans are warmer than ambient air, but cooler than surrounding sun-warmed surfaces.

Temperature can vary considerably in an environment. Living organisms have evolved sensory systems to detect and avoid excessive heat or cold: a behavior that is termed ‘thermotaxis’. In rare cases, animals use this ability to locate food sources in their environment. One example of such an adaptation is the female mosquito of the species Aedes aegypti. When a mosquito needs blood to produce her eggs, she becomes attracted to the body heat of warm-blooded hosts. But the range of temperatures that these mosquitoes prefer and the genes required for this behavior had not been been defined.

Now, Corfas and Vosshall have found that female Aedes aegypti are highly sensitive to differences in temperature, and are capable of heat-seeking in a range of environmental temperatures. Furthermore, by seeking out things that are warmer than their surroundings, while avoiding those that are cooler or much hotter than their host’s body temperatures, these mosquitoes tune their thermotaxis toward targets that resemble a human to feed upon.

Corfas and Vosshall also discovered that a protein called TRPA1 is required for this tuning of Aedes aegypti’s heat-seeking behavior. This protein is known to allow insects to detect chemical signals and regulate their own temperature, but it was not previously known that this protein was involved in mosquito thermotaxis. Mutant mosquitoes without the gene for TRPA1 failed to avoid high temperatures, which meant that they could no longer tell the difference between an overly hot target and a warm one that resembled their hosts.

Following on from this work, the next challenge will be to characterize all the genes, sensory organs, and neural circuits that drive mosquito heat-seeking behavior. These findings may in the future inform the design of the next generation of repellents and traps for the control of mosquito-borne diseases, such as dengue and yellow fever.

Introduction

Thermotaxis is a sensory-motor behavior that guides animals toward a preferred temperature. This type of sensory navigation allows animals to avoid environments of noxious cold and heat, with the goal of remaining in physiologically suitable ambient temperatures. For ectotherms, such as most insects, thermotaxis behavior is the primary method of thermoregulation. Terrestrial invertebrates are vulnerable to temperature extremes, facing the risk of desiccation at elevated temperatures, and rapid hypothermia at low temperatures. Therefore, mechanisms to detect environmental temperatures and trigger appropriate approach or avoidance behaviors are extremely important for their survival. For instance, adult Caenorhabditis elegans worms migrate preferentially toward a specific thermal environment determined by the temperature of their cultivation (Hedgecock and Russell, 1975; Mori and Ohshima, 1995). Adult Drosophila melanogaster flies prefer a narrow range of air temperatures around 24–25°C (Sayeed and Benzer, 1996; Hamada et al., 2008) and rapidly avoid air temperatures of ~31°C (Ni et al., 2013).

Interestingly, some hematophagous (blood-feeding) arthropods have evolved a specialized mode of thermotaxis to locate endothermic (warm-blooded) hosts. Such thermophilic behavior is seen in kissing bugs [Triatoma infestans (Flores and Lazzari, 1996) and Rhodnius prolixus (Schmitz et al., 2000)], the bedbug [Cimex lectularius (Rivnai, 1931)], the tick [Ixodes ricinus (Lees, 1948)], and many species of mosquito (Clements, 1999) including Ae. aegypti, a major tropical disease-vector (Bhatt et al., 2013). Female Ae. aegypti require a vertebrate blood meal for the production of eggs, and finding a suitable warm-blooded host is therefore an essential component of reproduction. Mosquitoes use a variety of physical and chemical senses to locate hosts in their environment (Cardé, 2015). When host-seeking, these animals become strongly attracted to inanimate warm objects, eagerly probing at them as if they were hosts (Howlett, 1910).

In nature, mosquitoes thermotax in a complex thermal landscape in which ambient air temperature, host body temperature, and surrounding surface temperatures can vary widely. For mosquitoes such as Ae. aegypti, host-seeking behavior can be activated by an increase in ambient carbon dioxide (CO2) (Majeed et al., 2014). This activation elicits flight activity (Eiras and Jepson, 1991; McMeniman et al., 2014) and results in an array of behaviors including attraction to visual stimuli (van Breugel et al., 2015) and host olfactory cues (Dekker et al., 2005; McMeniman et al., 2014), and landing on warm objects (Burgess, 1959; Eiras and Jepson, 1994; Kröber et al., 2010; Maekawa et al., 2011; McMeniman et al., 2014; van Breugel et al., 2015). Ae. aegypti flying in a wind tunnel can detect a warmed stimulus from a distance, eliciting attraction and thermotaxis (van Breugel et al., 2015).

What are the mechanisms by which animals detect thermal stimuli, and how might these be adapted for the specialized needs of heat-seeking female mosquitoes? Thermotaxis is typically initiated by thermosensitive neurons that sample environmental temperature to inform navigational decision-making. Such neurons must be equipped with molecular thermosensors capable of detecting and transducing thermal stimuli. Diverse molecular thermoreceptors have been identified in the animal kingdom, many of which are members of the transient receptor potential (TRP) superfamily of ion channels (Barbagallo and Garrity, 2015; Palkar et al., 2015). Different thermosensitive TRPs show distinct tuning spanning the thermal spectrum from noxious cold to noxious heat. Among these is TRPA1, which is a heat sensor in multiple insects, including the vinegar fly D. melanogaster and the malaria mosquito Anopheles gambiae (Hamada et al., 2008; Wang et al., 2009). Neurons in thermosensitive sensilla (Gingl et al., 2005) of An. gambiae female antennae express TRPA1 (Wang et al., 2009). In D. melanogaster, TRPA1 is expressed in internal thermosensors located in the brain, and DmelTRPA1-/- mutants fail to avoid high air temperature in a thermal gradient (Hamada et al., 2008). Interestingly, some snakes and vampire bats express thermosensitive TRP channels in organs used to sense infrared radiation from warm-blooded prey (Gracheva et al., 2010; 2011). This raises the possibility that AaegTRPA1 may be used by mosquitoes to find hosts. Recently, a structurally distinct insect thermosensor, Gr28b, was identified in D. melanogaster (Ni et al., 2013). Gr28b, a gustatory receptor paralog, is expressed in heat-sensitive neurons of D. melanogaster aristae and is an important component of thermotaxis during rapid avoidance of heat (Ni et al., 2013). It is also highly conserved among Drosophila species (McBride et al., 2007), and has a clear ortholog in Ae. aegypti, AaegGr19 (Ni et al., 2013). A functional role for these thermosensors has never been investigated in the mosquito.

Here, we use high-resolution quantitative assays to examine the behavioral strategies underlying mosquito heat-seeking behavior. Our results show that by seeking relative warmth and avoiding both relative cool and high temperatures, female mosquitoes selectively localized to thermal stimuli that approximate warm-blooded hosts. Using genome editing, we generated mutations in the candidate thermoreceptors, AaegTRPA1 and AaegGr19. We found that TRPA1 is required for tuning mosquito thermotaxis during host-seeking. AaegTRPA1-/- mutants lacked normal avoidance of thermal stimuli exceeding host body temperatures, resulting in a loss of preference for biologically relevant thermal stimuli that resemble hosts. This work is important because it identifies a key mechanism by which mosquitoes tune their thermosensory systems toward human body temperatures.

Results

We previously described an assay to model heat-seeking behavior in the laboratory by monitoring mosquitoes landing on a warmed Peltier element in the context of a cage supplemented with CO2 (Figure 1A,B) (McMeniman et al., 2014). This assay has the advantages that it is simple in design, produces robust behaviors, and enables the collection of data from large numbers of animals in a short experimental timeframe. Using this system, we can examine mosquito responses to diverse thermal stimuli and measure thermotaxis in different ambient temperature environments. We first needed to determine whether heat-seeking behavior habituates over multiple thermal stimulations. In our heat-seeking assay, Ae. aegypti mosquitoes reliably responded to 12 serial presentations of a 3-minute long 40°C stimulus over the course of more than 2.5 hr, with no evidence of habituation (Figure 1—figure supplement 1).

Ae. aegypti can feed on a variety of hosts (Clements, 1999; Tandon and Ray, 2000) with core body temperatures ranging from ~37°C (humans) to ~40–43°C (chickens) (Richards, 1971) (Figure 1C). It is unknown whether there are minimal or maximal temperature thresholds constraining mosquito heat-seeking, and whether responses to thermal stimuli depend on the background ambient temperature.

To investigate these questions, we measured attraction to thermal stimuli produced by heating the Peltier to temperatures ranging from ambient (set to 26°C in these experiments) to 60°C (Figure 1D, E). We found that mosquitoes were highly sensitive to thermal contrast and were attracted to stimuli 2.5°C above ambient (Figure 1D–F). Furthermore, mosquito occupancy on the Peltier increased monotonically with stimulus temperatures up to 40°C. However, for higher temperature stimuli, we observed a dramatic reduction in Peltier occupancy. A 50°C stimulus resulted in approximately half as many animals on the Peltier compared to a 40°C stimulus (Figure 1D–F). Stimuli of 55°C or greater resulted in occupancy rates indistinguishable from an ambient thermal stimulus (26°C) (Figure 1D–F). Spatial analysis of mosquito occupancy on or near the Peltier revealed that while mosquitoes were still attracted to high-temperature stimuli, they populated the area peripheral to the Peltier, and strongly avoided the Peltier itself for stimuli ≥ 55°C (Figure 1F).

Female mosquitoes searching for a warm-blooded host may be responding to the absolute temperature of a stimulus or may instead be evaluating relative warmth, defined as the differential between a stimulus and background ambient temperature. To investigate the thermotaxis strategies constituting mosquito heat-seeking behavior, we conducted experiments at three ambient temperatures: 21, 26, and 31°C (Figure 2A–F). We found that Peltier occupancy for stimuli 21–40°C depended on the differential between the Peltier and ambient temperature (Figures 2B,C), rather than the absolute temperature of the Peltier (Figure 2A). For example, at all ambient temperatures tested, a stimulus 5°C above ambient was sufficient to elicit significant heat-seeking, and elicited approximately half as much Peltier occupancy as a stimulus 10°C above ambient. On the other hand, heat-seeking to targets 50–55°C was inhibited at all ambient temperatures tested (Figures 2D,F), despite the fact that the temperature differential varied widely in these situations (Figure 2E).

The Article can be read in full in eLife  DOI

Posted: December 17, 2015

Up

Generation of Contractile Actomyosin Bundles Depends on Mechanosensitive Actin Filament Assembly and Disassembly


In a research published in eLife Sari Tojkander, Gergana Gateva, Amjad Husain, Ramaswamy Krishnan, Pekka Lappalainen ( University of Helsinki, Finland; Harvard Medical School, United States) writes that
Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells.

Muscle cells are the best-known example of a cell in the human body that can contract. These cells contain bundles of filaments made of proteins called actin and myosin, which can generate pulling forces. However, many other cells in the human body also rely on similar “contractile actomyosin bundles” to help them stick to each other, to maintain the correct shape or to migrate from one location to another. These bundles in the non-muscle cells are often called “ventral stress fibers”.

Ventral stress fibers develop from structures commonly referred to as “arcs”. Previous work has clearly established that ventral stress fibers are sensitive to mechanical forces. However, the underlying mechanism behind this process was not known, and it remained unclear how external forces could promote these actomyosin bundles to assemble, align and mature.

Tojkander et al. documented the formation of ventral stress fibers in migrating human cells grown in the laboratory. This revealed that pre-existing arcs fuse with each other to form thicker and more contractile actomyosin bundles. The formation of these bundles then pulls on the two ends of the stress fibers that are attached to sites on the edges of the cell.

Tojkander et al. also showed that this tension inactivates a protein called VASP, which is also found at these sites. Inactivating VASP inhibits the construction of actin filaments, which in turn stops the stress fibers from elongating and allows them to contract. Further experiments then revealed that ventral stress fibers are maintained and can even become thicker under a sustained pulling force. Conversely, stress fibers that were not under tension were decorated by proteins that promote the disassembly of actin filaments. This subsequently led to the disappearance of these fibers.

Future studies could now examine whether the newly identified pathway, which allows mechanical forces to control the assembly and alignment of stress fibers, is conserved in other cell-types. Furthermore, and because the assembly of such mechanosensitive actomyosin bundles is often defective in cancer cells, it will also be important to study this pathway’s significance in the context of cancer progression.

Introduction

Cell migration is essential for embryonic development, wound healing, immunological processes and cancer metastasis. Cell migration is driven by assembly and disassembly of protrusive and contractile actin filament structures. The force in protrusive actin filament structures, including lamellipodium and filopodia at the leading edge of cell, is generated through actin polymerization against the plasma membrane. In contractile actin filament bundles, such as stress fibers, the force is generated by sliding of bipolar myosin II bundles along actin filaments. Notably, whereas the assembly-mechanisms of protrusive actin filament structures are relatively well understood, general principles underlying the assembly of contractile actomyosin bundles have remained elusive (Pollard and Cooper, 2009; Bugyi and Carlier, 2010; Michelot and Drubin, 2011; Burridge and Wittchen, 2013).

The most prominent contractile actomyosin structures in most cultured non-muscle cells are stress fibers. Beyond cell migration, stress fibers guide adhesion, mechanotransduction, endothelial barrier integrity, myofibril assembly, and receptor clustering in T-lymphocytes (Burridge and Wittchen, 2013; Wong et al., 1983; Sanger et al., 2005; Tojkander et al., 2012; Yi et al., 2012). Due to their intrinsic properties, stress fibers have become an important model system for studying the general principles by which contractile actomyosin bundles are assembled in cells. Stress fibers can be divided into three main categories based on their protein compositions and interactions with focal adhesions (Small et al., 1998). Dorsal (radial) stress fibers are connected to focal adhesions at their distal ends and rise towards the dorsal surface of the cell at their proximal region (Hotulainen and Lappalainen, 2006). They elongate through vectorial actin polymerization at focal adhesions (i.e. coordinated polymerization of actin filaments, whose rapidly elongating barbed ends are facing the focal adhesion, is responsible for growth of dorsal stress fibers). These actin filament bundles do not contain myosin II, and dorsal stress fibers are thus unable to contract (Hotulainen and Lappalainen, 2006; Cramer et al., 1997; Tojkander et al., 2011; Oakes et al., 2012; Tee et al., 2015). However, dorsal stress fibers interact with contractile transverse arcs and link them to focal adhesions. Transverse arcs are curved actin bundles, which display periodic α-actinin – myosin II pattern and undergo retrograde flow towards the cell center in migrating cells. They are derived from α-actinin- and tropomyosin/myosin II- decorated actin filament populations nucleated at the lamellipodium of motile cells (Hotulainen and Lappalainen, 2006; Tojkander et al., 2011; Burnette et al., 2011; 2014). In fibroblasts and melanoma cells, filopodial actin bundles can be recycled for formation of transverse arc –like contractile actomyosin bundles (Nemethova et al., 2008; Anderson et al., 2008). Ventral stress fibers are defined as contractile actomyosin bundles, which are anchored to focal adhesions at their both ends. Despite their nomenclature, the central regions of ventral stress fibers can bend towards the dorsal surface of the lamellum (Hotulainen and Lappalainen, 2006; Schulze et al., 2014). Migrating cells display thick ventral stress fibers that are typically oriented perpendicularly to the direction of migration, and thinner ventral stress fibers that are often located at the cell rear or below the nucleus. At least the thick ventral stress fibers, which constitute the major force-generating actomyosin bundles in migrating cells, are derived from the pre-existing network of dorsal stress fibers and transverse arcs. However, the underlying mechanism has remained poorly understood (Burridge et al., 2013; Hotulainen and Lappalainen, 2006).

Stress fibers and focal adhesions are mechanosensitive structures. Stress fibers are typically present only in cells grown on rigid substrata and they disassemble upon cell detachment from the matrix (Mochitate et al., 1991; Discher et al., 2005). Furthermore, after applying fluid shear stress, stress fibers align along the orientation of flow direction in endothelial cells (Sato and Ohashi, 2005). Also focal adhesions develop only on rigid surfaces, and applying external tensile force promotes their enlargement (Chrzanowska-Wodnicka and Burridge, 1996; Pelham et al., 1999; Riveline et al., 2001). Focal adhesions contain several mechano-sensitive proteins, including talin, filamin and p130Cas, whose activities and interactions with other focal adhesion components can be modulated by forces of ~∼10–50 pN range (Sawada et al., 2006; del Rio et al., 2009; Ehrlicher et al., 2011). Furthermore, the protein compositions of focal adhesions are regulated by tension supplied by myosin II activity and external forces applied to the cell (Zaidel-Bar et al., 2007; Kuo et al., 2011; Schiller et al., 2011). Importantly, despite wealth of information concerning mechanosensitive focal adhesion proteins, possible effects of tensile forces on actin filament assembly at focal adhesions have remained elusive. Furthermore, the mechanisms by which tension contributes to the alignment of stress fibers and actin dynamics within these actomyosin bundles have not been reported.

Here we reveal that formation of mature contractile actin bundles from their precursors is a mechanosensitive process. We show that arc fusion during centripetal flow is accompanied by increased contractility that inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, thus insuring formation of ventral stress fibers. Conversely, activation of AMPK allows generation of contractile ventral stress fibers in cells growing on compliant matrix, where their formation is normally prevented. Furthermore, we provide evidence of mechanosensitive actin filament disassembly by ADF/cofilins during stress fiber assembly. These data provide support to a new mechanobiological model explaining the principles of assembly and alignment of ventral stress fibers in migrating cells.

Results

Transverse arcs fuse with each other during centripetal flow

Transverse arcs are generated from actin filament arrays at the lamellipodium –— lamella interface (Tojkander et al., 2011; Shemesh et al., 2009; Burnette et al., 2011). During their assembly, thin arcs associate with elongating dorsal stress fibers to form a spider-net -like structure (Figure 1—figure supplement 1A and 1B; Tojkander et al., 2011). This network, consisting of several non-contractile dorsal stress fibers and multiple thin arcs, flows towards the cell center and maturates to thick, contractile ventral stress fibers through a mechanism that has remained poorly understood (Hotulainen and Lappalainen, 2006). Interestingly, proper stress fiber network does not form in cells grown on compliant matrix (Discher et al., 2005; Prager-Khoutorsky et al., 2011), but whether the assembly of all above-mentioned stress fiber categories, or only a specific one, is mechanosensitive has not been reported. By plating U2OS cells on soft (0.5 kPa) and stiff (64 kPa) substrata, we revealed that dorsal stress fibers and arcs are also present in cells grown on compliant matrix. In contrast, ventral stress fiber assembly is compromised under these conditions (Figure 1A). While 89% of cells plated on 64 kPa matrix contained ventral stress fibers, only 10% of cells plated on 0.5 kPa matrix exhibited ventral stress fibers as defined by presence of straight, contractile actin bundles connected to focal adhesions at each end. Thus, generation of ventral stress fibers appears to be the mechanosensitive phase in the formation of the stress fiber network.

To reveal how ventral stress fibers are derived from arcs and to elucidate the mechanosensitive basis of this process, we examined the dynamics of the stress fiber network in U2OS cells, where all three stress fiber categories can be readily visualized by live-cell microscopy (Hotulainen and Lappalainen, 2006). We first followed this process by using GFP-calponin-3 (CaP3), which compared to other stress fiber components allows better visualization of thin arc precursors. Interestingly, live-imaging of GFP-CaP3 -transfected cells revealed that the thin arc precursors fused with each other to form thicker actomyosin bundles during their flow towards the cell center (Figure 1B; Figure 1—figure supplement 1B). Fusion appeared to often initiate at the sites where arcs were connected to elongating dorsal stress fibers (Figure 1C). Live-imaging of cells expressing CFP-α-actinin and YFP-tropomyosin-4 demonstrated that homotypic coalescence of tropomyosin-4/myosin II foci and α-actinin foci of adjacent arcs occurred during the fusion process in all observed cases (Figure 1D). Thus, thin arc precursors fuse with each other during centripetal flow to generate thicker actomyosin bundles, where the periodic α-actinin — myosin II pattern is retained.

Transverse arc fusion is accompanied by increased contractility of actomyosin bundles and alignment of distal focal adhesions

Traction force microscopy was applied to examine whether arc fusion during centripetal flow is accompanied by changes in their contractility. These experiments revealed that thick ventral stress fibers exhibit stronger traction forces to focal adhesions as compared to forces applied by dorsal stress fibers (Figure 2A and B), similarly to what was recently demonstrated with model-based traction force microscopy by Soine et al. (2015). Furthermore, spacing between individual CaP-3 foci, which co-localize with α-actinin in stress fibers (Small and Gimona, 1998), decreased as the arcs flowed towards the cell center and become thicker as detected both from several fixed samples and live-cell imaging experiments (representative examples are shown Figure 1—figure supplement 1C and D). This correlates well with the increased contractility of the structures (Aratyn-Schaus et al., 2011).

Transverse arcs are typically connected to several focal adhesion-attached dorsal stress fibers along their length (Hotulainen and Lappalainen, 2006). To elucidate how increased contractility of arcs affects the associated focal adhesions, we examined possible changes in adhesion alignment during the arc maturation process. These experiments revealed that the ‘distal’ focal adhesions, linked via dorsal stress fibers to the ends of the arc, turned and aligned along the direction of arc. In contrast, focal adhesions linked to the central region of the arc did not display similar alignment during the process. Alignment of ‘distal’ focal adhesions correlated with arc fusion, and was accompanied by enlargement of adhesions (Figure 2—figure supplement 1A and B). Thus, arc fusion during centripetal flow correlates with their increased contractility, consequent enlargement of distal focal adhesions and their alignment along the direction of the actomyosin bundle. Eventually, this leads to formation of a directed ventral stress fiber, containing one properly aligned large focal adhesion at its both ends.

Tension provided by myosin II inhibits vectorial actin polymerization at focal adhesions

Dorsal stress fibers elongate through actin polymerization at focal adhesions. In U2OS cells, this ‘vectorial’ actin polymerization promotes elongation of the actin filament bundle with a rate of ∼0.25 μm/min (Hotulainen and Lappalainen, 2006). In addition, focal adhesions may contain other actin filament populations that are not directly associated with vectorial actin polymerization and consequent elongation of dorsal stress fibers. This is because several tropomyosin isoforms, which are likely to decorate distinct actin filament populations, localize to focal adhesions (Tojkander et al., 2011) and because several proteins involved in actin polymerization regulate actin dynamics at focal adhesions (e.g. Hotulainen and Lappalainen, 2006; Skau et al., 2015). Furthermore, FRAP experiments performed at focal adhesions show rapid, uniform recovery of GFP-actin fluorescence (Videos 1 and 2; Figure 2—figure supplement 2), whereas FRAP experiments performed at dorsal stress fiber regions below focal adhesions exhibit treadmilling-like recovery that is indicative of vectorial actin polymerization (Hotulainen and Lappalainen, 2006; Tee et al., 2015).

Because contractility promotes focal adhesion enlargement and alignment during maturation of arcs to ventral stress fibers, we examined whether this process would be accompanied by alterations in vectorial actin polymerization at focal adhesions. Fluorescence-recovery-after-photobleaching (FRAP) was first applied to visualize the recovery of GFP-actin signal within actin filament bundles of dorsal and ventral stress fibers. Region of interest was chosen beneath focal adhesions to exclude other focal adhesion associated actin filament populations that are not directly involved in vectorial actin polymerization and elongation of stress fibers. As previously reported, elongation of a bright actin filament bundle (with a rate of ∼0,26 μm/min) from focal adhesions located at the distal ends of dorsal stress fibers was observed (Hotulainen and Lappalainen, 2006). Importantly, when a FRAP analysis was performed on a corresponding ventral stress fiber region, only very slow elongation (∼0,02 μm/min) of a bright actin filament bundle from the adhesion was observed. Instead, we mainly detected recovery of GFP-actin fluorescence evenly along the photobleached region (Figure 2C-E). As an alternative approach, we utilized photoactivatable (PA)-GFP-actin to follow its incorporation into dorsal and ventral stress fibers. In both cases, significant fraction of activated PA-GFP-actin remained at/close to focal adhesions, probably corresponding to actin filament pools associated with focal adhesions (Tojkander et al., 2011). Importantly, PA-GFP-actin displayed centripetal flow along the actin filament bundle from focal adhesions in dorsal stress fibers, while similar flow of PA-GFP-actin was not detected from focal adhesions located at the tips of ventral stress fibers (Figure 2F). Therefore, in contrast to dorsal stress fibers, ventral stress fibers do not elongate through vectorial actin polymerization at focal adhesions.

To elucidate whether inhibition of vectorial actin polymerization in focal adhesions at the tips of ventral stress fibers is dependent on tension applied by myosin II, we examined the morphology of the stress fiber network in cells treated with myosin light chain kinase (MLCK) inhibitor ML-7. This compound induced rapid disassembly of most contractile ventral stress fibers and transverse arcs, without affecting integrity of non-contractile dorsal stress fibers (Figure 2—figure supplement 3C). Importantly, dorsal stress fibers in cells treated for 2 h with ML-7 were ∼1.5 times longer compared to the ones in control cells (Figure 2—figure supplement 2D). Similarly, disruption of contractile stress fibers by ROCK inhibitor, Y27632, or by over-expression of dominant inactive Rif GTPase (Rif-TN), which prevents assembly of contractile arcs (Tojkander et al., 2011), led to formation of abnormally long dorsal stress fibers (Figure 2—figure supplement 3A and B, and Figure 2—figure supplement 4). Importantly, live-imaging of GFP-actin expressing cells revealed that the abnormally long dorsal stress in Rif-TN transfected cells continued to elongate throughout the entire observation period. During their uncontrolled elongation, the dorsal stress fibers of Rif-TN expressing cells occasionally bent or fused with another elongating dorsal stress fiber initiated from the opposite side of the cell (Figure 2—figure supplement 4A; Videos 3 and 4).

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Posted: December 16, 2015

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Protein Aggregates are Associated with Replicative Aging Without Compromising Protein Quality Control

Juha Saarikangas and Yves Barral

( Published On eLifeSciences )

Differentiation of cellular lineages is facilitated by asymmetric segregation of fate determinants between dividing cells. In budding yeast, various aging factors segregate to the aging (mother)-lineage, with poorly understood consequences. In this study, we show that yeast mother cells form a protein aggregate during early replicative aging that is maintained as a single, asymmetrically inherited deposit over the remaining lifespan. Surprisingly, deposit formation was not associated with stress or general decline in proteostasis. Rather, the deposit-containing cells displayed enhanced degradation of cytosolic proteasome substrates and unimpaired clearance of stress-induced protein aggregates. Deposit formation was dependent on Hsp42, which collected non-random client proteins of the Hsp104/Hsp70-refolding machinery, including the prion Sup35. Importantly, loss of Hsp42 resulted in symmetric inheritance of its constituents and prolonged the lifespan of the mother cell. Together, these data suggest that protein aggregation is an early aging-associated differentiation event in yeast, having a two-faceted role in organismal fitness.

Aging is a complex process. Studies involving a single-celled organism called budding yeast are commonly used to investigate the factors that contribute to aging. When these yeast cells divide, a small daughter cell buds out from a large mother cell. A mother cell has a limited lifespan and produces a finite number of daughter cells and then dies (a phenomenon referred to ‘replicative aging’). However, when a daughter cell forms, the daughter's age is reset to zero, giving it the full potential to produce new offspring.

Previous research on budding yeast has shown that damaged or aggregated proteins accumulate in the mother cells but not the daughter cells, and that this accumulation of proteins contributes to shortening the lifespan of the mother cell. Furthermore, protein aggregation has also been associated with a number of age-related diseases in humans, including neurodegenerative disorders such as Alzheimer's and Parkinson's disease. However, it remains unclear how cells respond to protein aggregation that occurs during aging.

Many studies that have previously investigated this question have relied on exposing cells to stressful conditions, such as high temperatures, in order to trigger proteins to aggregate. But now, Saarikangas and Barral have studied how proteins aggregate under normal, unstressed conditions in budding yeast as they age. The experiments revealed that most unstressed yeast cells develop a single deposit of aggregated proteins already during early aging. This age-associated structure proved to be a different type of response than the protein aggregation that occurs during stress.

Furthermore, the deposit did not form as a consequence of the cell having obvious problems with folding its proteins, nor did the deposit hinder cells from coping with stressful conditions that trigger protein misfolding. Rather, this deposit supported the ability of the cell to degrade defective proteins. This suggests that the deposit represents an early adaptive response to aging, which might consequently provide aged cells some advantage over their younger counterparts.

Saarikangas and Barral also found that this protein deposit was always retained in the mother cell and not passed onto the daughters at cell division. Further experiments showed that an enzyme called heat shock protein 42 was responsible for collecting target proteins and bring them together to form the single deposit. Reducing the levels of this enzyme prevented the deposit from forming and extended the lifespan of the mother cells. Thus, these findings suggest that mother cells collect harmful protein aggregates into a single deposit and prevent them from entering the daughter cells. Further work is needed to understand how the deposit is preferentially retained within the mother cell.

Introduction

Aging results in an increasing decline of the organism's fitness over time (Lopez-Otin et al., 2013). Remarkably, this process segregates asymmetrically during budding yeast division: the mother cell forms an aging lineage, whereas the daughters generated by these mothers rejuvenate to form eternal lineages, similar to the segregation of soma and germ lineages in metazoans. Such lineage separation requires that the inheritance of factors that promote aging, such as defective/deleterious organelles, proteins, and DNA, is asymmetric during cell division (Sinclair and Guarente, 1997; Aguilaniu et al., 2003; Erjavec et al., 2007; Henderson and Gottschling, 2008; Shcheprova et al., 2008; Liu et al., 2010; Zhou et al., 2011; Hughes and Gottschling, 2012; Higuchi et al., 2013; Clay et al., 2014; Denoth Lippuner et al., 2014; Henderson et al., 2014; Higuchi-Sanabria et al., 2014; Thayer et al., 2014; Katajisto et al., 2015). Therefore, how cells are able to recognize, sort, and coordinate the asymmetric segregation of aging factors and other fate determinants is an outstanding question in biology (Neumuller and Knoblich, 2009).

Protein aggregates and/or damaged proteins are a hallmark in the etiology of many human disorders associated with aging (Hartl et al., 2011; Wolff et al., 2014), and their presence correlates with aging of mitotically active yeast and drosophila stem cells (Aguilaniu et al., 2003; Erjavec et al., 2007; Bufalino et al., 2013; Coelho et al., 2013). Studies on budding yeast have shown a correlation between the accumulation of protein aggregates and replicative aging by demonstrating that Hsp104-mediated protein disaggregation is required for full replicative life span (Erjavec et al., 2007), and that over-expression of Mca1, which counteracts the formation of stress- and age-associated protein aggregates (Lee et al., 2010; Hill et al., 2014), extends the life span of yeast mother cells (Hill et al., 2014).

How cells respond to protein aggregation that occurs specifically during aging has remained elusive since most studies investigating the cellular responses to protein aggregation have relied on over-expression of non-native, aggregation prone proteins, proteostasis inhibitors, or other stressors, such as heat (Kaganovich et al., 2008; Liu et al., 2010; Specht et al., 2011; Zhou et al., 2011; Malinovska et al., 2012; Spokoini et al., 2012; Winkler et al., 2012; Escusa-Toret et al., 2013; Zhou et al., 2014). These studies have uncovered specific modes of cytosolic compartmentalization that take place when cells encounter proteotoxic stress. For example, cells stressed with heat respond by forming multiple protein aggregates (referred to as peripheral aggregates, stress foci, Q-bodies, or CytoQ) at the surface of the ER (Specht et al., 2011; Spokoini et al., 2012; Escusa-Toret et al., 2013; Miller et al., 2015; Zhou et al., 2014; Wallace et al., 2015). These structures, hereafter referred as Q-bodies, contain acutely misfolded proteins that are sorted between the nuclear and cytoplasmic degradation/deposit sites by the Hook family proteins Btn2 and Cur1 (Malinovska et al., 2012), and coalesce together by the aid of small heat shock proteins; Hsp42 in budding yeast (Specht et al., 2011; Escusa-Toret et al., 2013), and Hsp16 in fission yeast (Coelho et al., 2014). Simultaneously, Q-bodies are being rapidly resolved by the protein disaggregase Hsp104 (Parsell et al., 1994; Specht et al., 2011; Zhou et al., 2011; Spokoini et al., 2012; Escusa-Toret et al., 2013), together with other heat shock responsive chaperones such as Hsp70 and Hsp82 (Escusa-Toret et al., 2013). The formation of Q-bodies seems to aid stress survival, as the deletion of HSP42 resulted in defective tolerance of prolonged heat stress (Escusa-Toret et al., 2013). The asymmetric inheritance of Q-bodies by the mother cells is promoted by the geometry of the bud neck (Zhou et al., 2011), tethering to mitochondria (Zhou et al., 2014), and by actin cable-mediated retrograde transport, which is dependent of Hsp104 and Sir2 (Liu et al., 2010; Song et al., 2014). Notably, Sir2 is also a key player in processes that underlie the asymmetric segregation of damaged mitochondria (Higuchi et al., 2013) and the accumulation of extrachromosomal DNA circles (Sinclair and Guarente, 1997; Kaeberlein et al., 1999) to the aging mother cell.

Prolonged Q-body-inducing stress (heat or over-expression of thermolabile proteins) combined with proteasome inhibition can lead to the formation of a dynamically exchanging deposit of ubiquitylated proteins named the juxtanuclear quality compartment, JUNQ (Kaganovich et al., 2008; Escusa-Toret et al., 2013). This structure is regulated by the Upb3 deubiqutinase (Oling et al., 2014), by proteosomal activity (Andersson et al., 2013) and by lipid droplets (Moldavski et al., 2015), and it was also shown to appear during replicative aging (Oling et al., 2014). The faithful inheritance of this structure by the mother cell is dependent on its association with the nucleus (Spokoini et al., 2012). More recently, it was shown that the ‘JUNQ’ might actually reside inside the nucleus, and it was thus renamed as intranuclear quality control compartment, INQ (Miller et al., 2015). The JUNQ/INQ assembly is dependent on Btn2-aggregase (Miller et al., 2015), a protein also found to be involved in prion curing (Kryndushkin et al., 2008, 2012). Apart from the JUNQ/INQ structure, terminally aggregating proteins, such as the amyloidogenic prions Rnq1 and Ure2, were shown to partition to an non-dynamic, vacuole-associated deposit called the insoluble protein deposit IPOD (Kaganovich et al., 2008; Tyedmers et al., 2010b), which has remained less well characterized.

Despite this wealth of data, it remains unclear how these exogenous/stress-induced aggregation models relate to protein aggregation that takes place during physiological ‘healthy’ aging. Particularly, it is unclear why/how protein aggregates arise during aging, how are they segregated during cell division and, importantly, what is their consequence to the protein quality control of the aging cell, as well as to the aging process itself. To illuminate these aspects, we probed the role of protein aggregation during unperturbed replicative aging. Our findings indicate that protein aggregation is a prevalent and highly coordinated event of early aging and is not solely associated with proteostasis deterioration. Instead, we provide evidence that age-associated protein aggregation may initially benefit the cytosolic protein quality control, but eventually becomes involved with age-associated loss of fitness.

Results

Formation of a protein deposit during early replicative aging

To address the role of protein aggregation in unperturbed, physiological aging, we analyzed microscopically the replicative age-associated protein aggregation landscape in budding yeast by visualizing different chaperone proteins that mark aberrantly folded and aggregated proteins. By employing the Mother Enrichment Program (MEP) (Lindstrom and Gottschling, 2009) (Figure 1—figure supplement 1A), we harvested cells of different age and first analyzed the localization of endogenous GFP-tagged protein-disaggregase Hsp104 (Parsell et al., 1994; Glover and Lindquist, 1998), a broad sensor for protein aggregates (Figure 1A, Haslberger et al., 2010). Interestingly, we found many cells displaying an aggregate (typically a single bright Hsp104-labeled focus) and this portion increased in a progressive, age-dependent manner such that >80% of cells that had undergone more than 6 divisions displayed such a structure (Figure 1A,B), as previously reported (Aguilaniu et al., 2003; Erjavec et al., 2007). Co-localization analysis with Hsp104 demonstrated that the Hsp70 proteins Ssa1 and Ssa2, the small heat shock protein Hsp42, and the Hsp40 protein Ydj1 readily localized to these aggregates, the Hsp26 was found to be enriched in only 15% of Hsp104-labeled foci, while no accumulation of Hsp40 protein Sis1 or the Hsp90 protein Hsp82 was detected (Figure 1C, Figure 1—figure supplement 2A–C). Importantly, age-dependent appearance of these structures was also detected in diploid cells, in other strain backgrounds (W303), independently of the MEP procedure, and when different fluorophores where used for tagging Hsp104 (Figure 1—figure supplement 2D–H), indicating that their formation represents a general, age-dependent phenomenon of budding yeast cells.

This article was published November 6, 2015 Cite as eLife 2015;4:e06197

The article can be read in full here : DOI

The Authors are from Eidgenössische Technische Hochschule Zürich, Switzerland

Posted on: November 11, 2015

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FAM150A and FAM150B are Activating Ligands for Anaplastic Lymphoma Kinase

In a recent research published in eLife  J, , , , , , , , , , , ,

Cells have receptor proteins on their surface that enable them to detect changes in their environment and communicate with other cells. Signal molecules bind to a segment of the receptor called the extracellular domain that faces out from the cell. This can result in the activation of another domain in the receptor that is just inside the cell, which, in turn, activates signaling pathways that relay the information around the cell.

However, these communication systems are often disrupted in cancer cells. This helps the cells to override the strict growth controls imposed upon them by other (healthy) cells in the body. The gene that encodes a receptor protein called Anaplastic Lymphoma Kinase (or ALK for short) is often mutated in some types of human cancer so that the protein is always active. However, we still do not know what signal molecules bind to the ALK protein to activate it in normal cells.

Guan, Umapathy et al. used a variety of cell biology and biochemical techniques to study the role of ALK. The experiments show that when either of two proteins called FAM150A and FAM150B are produced in rat nerve cells alongside ALK, the nerve cells rapidly respond and form outgrowths. Experiments using cancer cells derived from human nerve cells also yielded similar results. Guan, Umapathy et al. found that the extracellular domain of ALK can physically interact with FAM150A and FAM150B.

The eyes of fruit flies that had been genetically modified to produce the human ALK protein alongside either FAM150A or FAM150B grew more than normal, giving the eyes an abnormal "rough" appearance. Further experiments showed that FAM150A and FAM150B are also able to increase the level of activation of an ALK mutant protein that is already active. Therefore, in future, the development of drugs that stop FAM150A and FAM150B from binding to ALK may be useful for treating cancers that are driven by high levels of ALK activity. Many challenging questions lie ahead to better understand how FAM150A and FAM150B interact with ALK.

Introduction

Activation of anaplastic lymphoma kinase (ALK) is commonly due to fusion of the ALK kinase domain with a dimerization partner that drives activation, however, ALK activation also occurs in the context of the full length receptor, for example, as activating point mutations in neuroblastoma (Maris et al., 2007; Carén et al., 2008; Chen et al., 2008; George et al., 2008; Janoueix-Lerosey et al., 2008; Mosseé et al., 2008; Hallberg and Palmer, 2013). In many additional tumor types ALK overexpression and activation has been described, and it is unclear whether this is dependent on the activity of a ligand (Hallberg and Palmer 2013). The ALK receptors in both Drosophila and Caenorhabditis elegans have well defined ligands – Jeb (Englund et al., 2003; Lee et al., 2003; Stute et al., 2004) and HEN-1 (Ishihara et al., 2002), respectively. In contrast, vertebrate ALK has long been considered as an orphan receptor.

The human ALK locus encodes a classical receptor tyrosine kinase (RTK) comprising a unique extracellular ligand-binding domain, a transmembrane domain and an intracellular tyrosine kinase domain (Hallberg and Palmer, 2013). The extracellular portion of ALK which contains two MAM domains (named after meprin, A-5 protein and receptor protein tyrosine phosphatase μ), a glycine-rich region (GR) and a LDLa domain, is unique among the RTKs. ALK, and the related leukocyte tyrosine kinase (LTK) RTK, share kinase domain similarities as well as a GR in the membrane proximal portion of their extracellular domains (ECDs) (Iwahara et al., 1997; Morris et al., 1997). Recent screening of the extracellular proteome identified two novel secreted proteins as ligands for LTK – family with sequence similarity 150A (FAM150A) and family with sequence similarity 150B (FAM150B). Both bind to the ECD of the receptor leading to activation of downstream signaling in cell culture models (Zhang et al., 2014). FAM150A and FAM150B are unique, displaying homology only with one another but not with any other proteins in mammals (Zhang et al., 2014). Furthermore, we found the reported strong expression of FAM150B in the human adrenal gland (Zhang et al., 2014) intriguing, given the role of ALK in neuroblastoma.

Here we report the identification of FAM150A and FAM150B as potent ligands for human ALK. We investigated ALK activation by FAM150A and FAM150B proteins in PC12 cell neurite outgrowth assays where we observed a strong activation of ALK signaling. Conditioned medium containing either FAM150A or FAM150B was able to activate endogenous ALK signaling in neuroblastoma cells. We also employed the model organism Drosophila melanogaster as a readout for activation of ALK by FAM150A and FAM150B, showing that FAM150 proteins are able to robustly drive human ALK activation when ectopically coexpressed in the fly. FAM150A and FAM150B bind to the ECD of ALK and, in addition to activation of wild-type ALK, are able to drive ‘superactivation’ of activated ALK mutants from neuroblastoma. The GR of the ALK receptor ECD is important for FAM150 activation, and monoclonal antibodies (mAb) recognizing the GR of ALK are able to inhibit activation of ALK by FAM150A. In conclusion, our data show that ALK is robustly activated by FAM150A/B finally providing an answer to the identity of the elusive ligands for this RTK.

Results and discussion

ALK and the related LTK share similarity in their membrane proximal ECD in the form of a glycine-rich domain that is ∼250 amino acids in length (Figure 1A, GR depicted in grey). This domain contains multiple runs of up to eight glycine residues, and is unique to ALK and LTK within the human genome. The importance of the GR in ALK has been highlighted in Drosophila studies, where four independent point mutations leading to exchange of single glycine residues result in complete loss of function in vivo (Englund et al., 2003) (Figure 1—figure supplement 1). The similarity between ALK and LTK within the GR is ∼70%, with amino acid identity of 55%, containing a total of 51 conserved glycine residues (Figure 1B). Given this similarity, and the important role of the glycine-rich domain for function in Drosophila, we hypothesized that FAM150A and FAM150B, which were recently reported as ligands for LTK (Zhang et al., 2014) may act as ligands for ALK.

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Posted on : November 26, 2015

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The Endothelial Cells Experiment will Fly to ISS This Year to See How They Work in Space

Title Endothelial cells. Released 26/06/2015 5:50 pm. Copyright Scuola Superiore Sant’Anna, Pisa, Italy


Components of human endothelial cells stained for identification. In green is the ‘actin’ protein that allows the cells to move, adhere, divide and react to stimuli. In blue are the cell nuclei containing DNA.

The Endothelial Cells experiment will fly to the International Space Station this year to understand how the cells that line our blood vessels react to weightlessness. Endothelial cells contain our blood and contract or expand our blood vessels as needed, regulating the flow of blood to our organs.

Blood flow changes in space because gravity no longer pulls blood towards astronauts’ feet. By understanding the underlying adaptive mechanisms of how our bodies respond to weightlessness, this experiment aims to develop methods to help astronauts in space while showing possibilities for people on Earth – our endothelial cells become less effective with age – to live longer and healthier lives.

Cultured human endothelial cells will be grown in space in ESA’s Kubik incubator for two weeks and then ‘freeze’ them chemically for analysis back on Earth.

For the team behind this experiment getting the experiment setup to work in space was challenging. “What is routine in laboratory is difficult in space” explains project leader Debora Angeloni from Scuola Superiore Sant’Anna (one of the three Universities of Pisa, Italy), “in space we have less samples to work with and the experiment needs to be self-contained.”

The final experiment sits in the palm of your hand and is fully-automated and controlled electronically without the need to use up precious astronaut time.

“We expect the cells to express different genes, and to attach and move differently due to their trip in space. Among other things, the red-stained actin in this photo taken in preparation in our laboratory on Earth will be compared to the samples when they return from space.”

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Posted on: November 22, 2015

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A Forward Genetic Screen Reveals Novel Independent Regulators of ULBP1, an Activating Ligand for Natural Killer Cells

In a research paper, published in Benjamin G Gowen, Bryan Chim, Caleb D Marceau, Trever T Greene, Patrick Burr, Jeanmarie R Gonzalez, Charles R Hesser, Peter A Dietzen, Teal Russell, Alexandre Iannello, Laurent Coscoy, Charles L Sentman, Jan E Carette, Stefan A Muljo, David H Raulet writes that recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger.

Cancer is caused by a series of mutations that result in uncontrolled cell growth and division. Yet, the body's immune system can often detect and destroy abnormal cells before they cause tumors and disease. Natural killer cells are part of the immune system and have receptors on their surface that allow them to tell the difference between healthy host cells and host cells that are stressed or abnormal. Some of these receptors activate the natural killer cells when they bind to their target molecules. Other receptors have the opposite effect and inhibit the natural killer cells. Activation occurs when the signaling from the activating receptors is stronger than the signals from the inhibitory receptors.

One of the well-studied activating receptors recognizes a number of proteins and molecules that are produced by abnormal or tumor cells, including a protein called ULBP1. This protein is absent from the surface of healthy cells but is found in abundance on tumor cells. However, it is still not clear what drives tumor cells to produce ULBP1 (or other molecules) that are recognized by natural killer cell receptors.

Now, Gowen et al. report on a genetic screen that has revealed numerous genes that regulate the levels of ULBP1 in human cells. Many of these genes had independent effects that when added together accounted for most of the ULBP1 present on the cell surface.

Gowen et al. then explored some of the ‘regulators’ encoded by these genes in more detail. One called ATF4, which had previously been linked to stress responses, was shown to increase the expression of the gene for ULBP1 in cancer cells. Another regulator called RBM4 instead acted in a different way and at a later stage in ULBP1 production.

All together, these findings offer insight into the stress pathways that alert the immune system to abnormal cells. The next challenge will be investigating how these pathways might be exploited for cancer immunotherapy.

Introduction

Natural killer (NK) cells are lymphocytes of the innate immune system that play a critical role in limiting tumor growth (Vivier et al., 2011; Marcus et al., 2014; Mittal et al., 2014). NK cell activation is controlled by a balance of signals from activating and inhibitory receptors, which recognize cognate ligands expressed by potential target cells (Vivier et al., 2011; Shifrin et al., 2014). One of the best-studied NK-activating receptors is NKG2D, which is also expressed on certain subsets of T cells (Raulet, 2003). Engagement of NKG2D by its ligands displayed on a target cell membrane leads to NK cell activation, cytokine secretion, and lysis of target cells, such as tumor cells.

NKG2D recognizes a family of ligands that are structurally similar to MHC Class I proteins. Humans express up to eight NKG2D ligands (ULBP1-6, MICA, and MICB), and mice express 5–6 different ligands, depending on the strain (RAE-1α-ε, H60a-c, and MULT1) (Raulet et al., 2013). Healthy cells typically do not display NKG2D ligands on their surface and are thus poor targets for NKG2D-mediated lysis by NK cells. However, cellular stresses associated with transformation, viral infection, or other danger to the host cause the upregulation of NKG2D ligand expression (Raulet et al., 2013). Primary tumors and cancer cell lines frequently express one or more NKG2D ligand, and NKG2D expression is important for the control of tumors in vivo in models of spontaneous cancer (Guerra et al., 2008).

Tumors arise despite the tumor-suppressive effects of the immune system, and some tumors show evidence of adaptation to escape immune control (Schreiber et al., 2011). In the case of NKG2D-mediated tumor recognition, published results suggest that one mechanism of tumor immune evasion is the loss or decreased expression of NKG2D ligands (Guerra et al., 2008; McGilvray et al., 2009). In other cases, tumors progress despite sustained expression of NKG2D ligands (Vetter et al., 2002; Guerra et al., 2008; McGilvray et al., 2009; Hilpert et al., 2012). The evidence as a whole suggests that upregulation of NKG2D ligands on early stage tumor cells is part of a host defense mechanism, but that the immune response subsequently applies selective pressure for tumors that have either extinguished expression of NKG2D ligands or have activated immune suppressive mechanisms (Raulet and Guerra, 2009). Therefore, identifying factors and pathways that regulate NKG2D ligands will improve our understanding of the cellular properties used by the immune system to define unwanted cells and will also help reveal how tumors evade the corresponding immune responses.

Prior investigations have identified regulators of NKG2D ligands using a candidate approach based on the roles of these regulators in known stress pathways. Such approaches have implicated the DNA damage response pathway (Gasser et al., 2005), heat shock (Venkataraman et al., 2007; Nice et al., 2009), hyperproliferation (Jung et al., 2012), and pattern recognition receptors (Hamerman et al., 2004), among others, in the regulation of one or more NKG2D ligands. However, these pathways do not account fully for expression of ligands in tumor cells, since inhibiting them may decrease ligand expression but typically does not abrogate it. For example, the DNA damage response is active in many cancer cells and tumor cell lines, but inhibiting that pathway only partially inhibits ligand expression (Gasser et al., 2005; Gasser and Raulet, 2006; Soriani et al., 2014). Similarly, hyperproliferation can drive NKG2D ligand expression, but blocking proliferation does not completely eliminate NKG2D ligand expression by tumor cell lines (Jung et al., 2012). These findings suggest that unidentified molecular cues in tumor cells also initiate the expression of NKG2D ligands, allowing potentially dangerous tumor cells to be distinguished from normal cells. Identifying those cues, especially for human NKG2D ligands, is important for understanding the biological regulation of NKG2D ligands and devising approaches for immunotherapy based on that knowledge.

To identify novel drivers of NKG2D ligand expression, we performed a genome-wide loss-of-expression mutant screen in the tumor-derived human cell line HAP1 (Carette et al., 2009; Carette et al., 2011) and used CRISPR/Cas9 gene targeting methodology for confirmation of the hits and extension of the results. The results reveal previously unknown regulators for NKG2D ligands, provide evidence for selectivity of the regulators for specific ligands, and support the cooperation of different stress pathways in the regulation of one such ligand.

Results

A genome-wide screen to identify novel drivers of ULBP1 expression

Many tumors and cancer cell lines express multiple NKG2D ligands, possibly due to ongoing stress responses associated with the transformed state (Raulet et al., 2013). To identify novel drivers of human NKG2D ligand expression in transformed cells, we employed a retroviral gene-trap mutagenesis screen using the near-haploid human cell line HAP1 (Figure 1) (Carette et al., 2009; Carette et al., 2011). Like many cell lines, HAP1 cells express multiple NKG2D ligands (Figure 1—figure supplement 1). We chose to screen for drivers of ULBP1 expression because it showed the brightest staining on HAP1 cells, making it particularly amenable to our loss-of-expression screen. Following mutagenesis, we selected for mutants with decreased expression of ULBP1 but intact expression of the unrelated GPI-anchored protein CD55 (Figure 1A). Selection of CD55+ cells was used to reduce the fraction of selected cells that had lost ULBP1 expression due to mutations that alter cell surface expression of all proteins or of all GPI-linked proteins. In the first round of selection, we depleted ULBP1high cells from the mutant cell population using magnetic bead-based depletion of cells labeled with a ULBP1 antibody. After briefly expanding the selected cells, we used flow cytometry to further select for ULBP1lowCD55+ cells. Figure 1B shows ULBP1 and CD55 expression on WT and post-selection HAP1 cells.

The article can be read in full DOI

The Authors are of : University of California, Berkeley, United States; National Institute of Allergy and Infectious Diseases, United States; Stanford University School of Medicine, United States; Dartmouth Geisel School of Medicine, United States

Posted on: November 20, 2015

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A Molecular Mechanism of Mitotic Centrosome Assembly in Drosophila

Paul T Conduit, Jennifer H Richens, Alan Wainman, James Holder, Catarina C Vicente, Metta B Pratt, Carly I Dix, Zsofia A Novak, Ian M Dobbie, Lothar Schermelleh, Jordan W Raff has published a research paper in eLife  explaining that centrosomes comprise a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, but it is unclear how this occurs. In this study, we show that the centriole protein Asl initiates the recruitment of DSpd-2 and Cnn to mother centrioles; both proteins then assemble into co-dependent scaffold-like structures that spread outwards from the mother centriole and recruit most, if not all, other PCM components. In the absence of either DSpd-2 or Cnn, mitotic PCM assembly is diminished; in the absence of both proteins, it appears to be abolished. We show that DSpd-2 helps incorporate Cnn into the PCM and that Cnn then helps maintain DSpd-2 within the PCM, creating a positive feedback loop that promotes robust PCM expansion around the mother centriole during mitosis. These observations suggest a surprisingly simple mechanism of mitotic PCM assembly in flies.

Long protein filaments called microtubules perform a range of roles inside cells—for example, they give the cell its shape and help to divide its genetic material during cell division. In animal cells, microtubules emerge from structures called centrosomes. These contain two cylindrical structures called centrioles that are surrounded by a matrix of pericentriolar material made from several hundred different proteins. Problems with centrosomes have been linked to several disorders, including cancer.

In the fruit fly Drosophila, it was long thought that the pericentriolar material assembles on an underlying ‘scaffold’, the composition of which had remained unclear. A protein called Centrosomin was a good candidate molecule, as it is required to maintain the proper structure of the pericentriolar material. In addition, Centrosomin molecules continuously spread away from the centrioles into the matrix providing a clear centriole–matrix connection. However, if Centrosomin is not present in a cell, some protein is still recruited around the centrioles. Conduit et al. therefore suspected that Centrosomin works together with another protein to build the scaffold.

Conduit et al. used super-resolution microscopy to observe the behaviour of several proteins, thought most likely to help Centrosomin to form the scaffold. Only one, called DSpd-2, builds outwards from the centrioles like Centrosomin. Genetic tests showed that both Centrosomin and DSpd-2 are important for the other proteins to localize to the pericentriolar material. If one of either Centrosomin or DSpd-2 is missing from the cell, reduced amounts of protein are recruited around the centrioles but the matrix still partially forms. Without both proteins, however, the matrix does not form at all.

Conduit et al. found that a third protein helps to recruit Centrosomin and DSpd-2 to the older of the two centrioles (also known as the mother centriole). DSpd-2 then draws in more Centrosomin. As Centrosomin helps to hold the DSpd-2 proteins in the pericentriolar material, this enables even more Centrosomin to be recruited, and so forms a positive feedback loop that helps the scaffold to continue growing.

The findings of Conduit et al. provide a simple mechanism for building the scaffold that supports the formation of the centrosome in the fruit fly Drosophila. Whether a similar mechanism is used to construct centrosomes in other species remains to be investigated.

Introduction

Centrosomes help regulate many cell processes, including cell shape, cell polarity, and cell division (Doxsey et al., 2005; Bettencourt-Dias and Glover, 2007), and centrosome defects have been implicated in several human pathologies (Nigg and Raff, 2009; Zyss and Gergely, 2009). Centrosomes are the major microtubule (MT)-organising centres (MTOCs) in many animal cells. They form when centrioles assemble a matrix of pericentriolar material (PCM) around themselves. Several hundred proteins are concentrated in the PCM, including many MT-organising proteins, cell-cycle regulators, and checkpoint and signalling proteins (Müller et al., 2010); thus, the centrosome appears to function as an important co-ordination centre in the cell (Doxsey et al., 2005). Although centrioles usually organize only small amounts of PCM in interphase cells, the PCM expands dramatically as cells prepare to enter mitosis—a process termed centrosome maturation (Khodjakov and Rieder, 1999).

Many proteins have been implicated in mitotic PCM assembly. These include (1) centriole-associated proteins, such as ‘Asl/Cep152’ (Bonaccorsi et al., 1998; Varmark et al., 2007; Dzhindzhev et al., 2010) and ‘Sas-4/CPAP’ (Cho et al., 2006; Gopalakrishnan et al., 2011), (2) proteins that have a centriole-associated fraction and a fraction that spreads out into the PCM, such as ‘Pericentrin/D-PLP’ (Martinez-Campos et al., 2004; Zimmerman et al., 2004; Fu and Glover, 2012; Lawo et al., 2012; Mennella et al., 2012) and ‘DSpd-2/Cep192’ (Pelletier et al., 2004; Dix and Raff, 2007; Gomez-Ferreria et al., 2007; Giansanti et al., 2008; Zhu et al., 2008; Joukov et al., 2010; Decker et al., 2011; Joukov et al., 2014), (3) proteins that reside in the PCM, such as ‘Cnn/Cdk5Rap2’ (Megraw et al., 1999; Lucas and Raff, 2007; Fong et al., 2008; Barr et al., 2010; Conduit et al., 2010), ‘DGp71WD/NEDD1’ (Haren et al., 2006, 2009; Lüders et al., 2006; Manning et al., 2010), and ‘γ-tubulin’ (Sunkel et al., 1995; Hannak et al., 2002), and (4) mitotic protein kinases, such as ‘Polo/Plk1’ and ‘Aurora A’ (Barr and Gergely, 2007; Petronczki et al., 2008). In recent super-resolution microscopy studies, several of these proteins appeared to be highly organized around interphase centrioles, but the organisation of proteins within the extended mitotic PCM was much less apparent (Fu and Glover, 2012; Lawo et al., 2012; Mennella et al., 2012; Sonnen et al., 2012).

It has long been thought that the mitotic PCM is assembled on an underlying scaffold structure (Dictenberg et al., 1998; Schnackenberg et al., 1998). We recently showed that Drosophila Centrosomin (Cnn) can form such a scaffold around centrioles and that this scaffold is assembled from the inside out (Conduit et al., 2014): Cnn molecules continuously incorporate into the scaffold around the centrioles and the scaffold then fluxes slowly outward, away from the centrioles. This inside out assembly mechanism could be important, as it potentially allows the assembly of the mitotic PCM to be regulated by the centrioles.

Many PCM proteins, however, can be recruited to mitotic centrosomes in the absence of Cnn, albeit at reduced levels (Lucas and Raff, 2007), suggesting that at least one other protein must be able to form a scaffold around centrioles that can recruit other PCM components. We reasoned that such a scaffold might also be assembled from the inside out. To identify such a protein(s), we analyzed the dynamic behaviour of the eight Drosophila centrosomal proteins that, in addition to Cnn, have been most strongly implicated in mitotic PCM recruitment: Asl, Sas-4, D-PLP, DSpd-2, γ-tubulin, DGp71WD, Polo, and Aurora A. We found that only DSpd-2 behaves like Cnn, as it incorporates into the PCM close to the centrioles and then spreads slowly outward to form a scaffold-like structure that recruits other PCM components. Importantly, in the absence of either DSpd-2 or Cnn, PCM recruitment is diminished, but in the absence of both proteins, it is abolished. We show that Asl appears to initiate the recruitment of DSpd-2 and Cnn exclusively to the mother centrioles; DSpd-2 then helps to recruit more Cnn, while Cnn helps to maintain DSpd-2 within the PCM, thus creating a positive feedback loop that promotes the scaffold assembly. Thus, mitotic PCM assembly appears to be a surprisingly simple process in flies: Asl initiates the recruitment of Spd-2 and Cnn to mother centrioles, and these proteins then assemble into scaffolds that spread out from the mother centriole and form a platform upon which most, if not all, other PCM proteins ultimately assemble.

The article can be read in full DOI

The authors are from University of Oxford, United Kingdom; Medical Research Council Laboratory of Molecular Biology, United Kingdom

Posted on: November 12, 2015

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NOCA-1 Functions with γ-Tubulin and in Parallel to Patronin to Assemble Non-centrosomal Microtubule Arrays in C. elegans

Shaohe Wang, Di Wu, Sophie Quintin, Rebecca A Green, Dhanya K Cheerambathur, Stacy D Ochoa, Arshad Desai, Karen Oegema of University of California, San Diego, United States; Université de Strasbourg, France; Institut Clinique de la Souris, France, writes in a recent study published in eLifeSciences, Non-centrosomal microtubule arrays assemble in differentiated tissues to perform mechanical and transport-based functions. In this study, we identify Caenorhabditis elegans NOCA-1 as a protein with homology to vertebrate ninein. NOCA-1 contributes to the assembly of non-centrosomal microtubule arrays in multiple tissues. In the larval epidermis, NOCA-1 functions redundantly with the minus end protection factor Patronin/PTRN-1 to assemble a circumferential microtubule array essential for worm growth and morphogenesis. Controlled degradation of a γ-tubulin complex subunit in this tissue revealed that γ-tubulin acts with NOCA-1 in parallel to Patronin/PTRN-1. In the germline, NOCA-1 and γ-tubulin co-localize at the cell surface, and inhibiting either leads to a microtubule assembly defect. γ-tubulin targets independently of NOCA-1, but NOCA-1 targeting requires γ-tubulin when a non-essential putatively palmitoylated cysteine is mutated. These results show that NOCA-1 acts with γ-tubulin to assemble non-centrosomal arrays in multiple tissues and highlight functional overlap between the ninein and Patronin protein families.

Microtubules are hollow, rigid filaments that are found in the cells of animals and other eukaryotes. These filaments are built from smaller building blocks called tubulin heterodimers; and in dividing animal cells, they mainly emerge from structures called centrosomes. When a cell is dividing, arrays of microtubules that originate from centrosomes help assemble the spindle-like structure that segregates the chromosomes.

Many non-dividing or specialized cells—including neurons, skin cells and muscle fibers—assemble other arrays of microtubules that do not emerge from centrosomes, but nevertheless perform a variety of structural, mechanical and transport-based roles. Compared to the centrosomal arrays, much less is known about how these non-centrosomal microtubules are assembled.

A vertebrate protein called ‘ninein’ had previously been shown to be involved in anchoring microtubules at centrosomes. Ninein can change its localization from centrosomes to the cell surface in mammalian skin cells, suggesting that it might also have a role in assembling the peripheral microtubule arrays that are found in these cells. Now, Wang et al. have identified a protein from worms called NOCA-1, which contains a region similar to the part of ninein that was previously shown to be needed to anchor microtubules at centrosomes.

The experiments show that NOCA-1 guides the assembly of non-centrosomal microtubule arrays in multiple tissues in C. elegans worms. This includes in the outer layer of the worm's larvae, which is similar to mammalian skin. The results also highlight that NOCA-1 performs many of the same roles as a member of the Patronin family of proteins called PTRN-1, which interacts with the ‘minus’ end of a microtubule to prevent the microtubule from breaking apart.

Wang et al. also found that NOCA-1 works with another protein called γ-tubulin, which helps new microtubules to form and also interacts with microtubule minus ends. In contrast, PTRN-1 works independently of γ-tubulin. This suggests that NOCA-1 works together with γ-tubulin to protect new microtubule ends or promote their assembly, a role similar to what has been proposed for Patronin family proteins. Overall, Wang et al.'s results highlight the importance of ninein-related proteins in the assembly of non-centrosomal microtubule arrays and suggest overlapping roles for the ninein and Patronin families of proteins.

Differentiated cells assemble non-centrosomal microtubule arrays to perform structural, mechanical, and transport-based functions (Keating and Borisy, 1999; Bartolini and Gundersen, 2006). Examples include the neuronal microtubule arrays that structure axons and dendritic arbors (Kuijpers and Hoogenraad, 2011), longitudinal arrays of parallel microtubules in syncytial myotubes (Warren, 1974; Tassin et al., 1985), and non-centrosomal arrays in epithelial cells (Keating and Borisy, 1999; Bartolini and Gundersen, 2006). In simple epithelia, cells build arrays of parallel microtubules that run along their apical–basal axis (Keating and Borisy, 1999; Bartolini and Gundersen, 2006; Brodu et al., 2010; Feldman and Priess, 2012), whereas desmosomal cell–cell junctions organize microtubule arrays that form around the periphery of stratified epithelial cells in mouse skin (Lechler and Fuchs, 2007; Sumigray et al., 2012).

The radial organization of centrosomal arrays arises from the fact that microtubules are nucleated, and their nascent minus ends capped and anchored, by centrosomally targeted protein complexes. Similarly, assembly of non-centrosomal microtubule arrays is likely to involve targeting of microtubule nucleating, as well as minus-end protection and/or anchoring factors, to non-centrosomal sites. Important current goals include identifying the factors that control the assembly of non-centrosomal arrays and determining the extent of overlap between the mechanisms utilized at centrosomes and non-centrosomal sites in different tissues.

Complexes containing γ-tubulin, a specialized tubulin isoform implicated in microtubule nucleation (Zheng et al., 1995; Oegema et al., 1999; Kollman et al., 2011), are thought to contribute to the assembly of both centrosomal and non-centrosomal arrays. During the differentiation of Drosophila tracheal epithelial cells, both γ-tubulin complexes, and the center of microtubule nucleation in regrowth experiments, transition from centrosomes to the apical cell surface (Brodu et al., 2010). In Caenorhabditis elegans, γ-tubulin is also targeted to the cell surface in the embryonic epidermis and germline, and the apical cell surface in the intestinal epithelium (Zhou et al., 2009; Fridolfsson and Starr, 2010; Feldman and Priess, 2012).

Ninein is a large coiled-coil protein that localizes to the sub-distal appendages of mother centrioles (Mogensen et al., 2000), where it is thought to anchor centrosomal microtubules (Dammermann and Merdes, 2002; Delgehyr et al., 2005). During the differentiation of mouse cochlear epithelial cells, ninein re-localizes from centrosomes to the apical surface (Mogensen et al., 2000; Moss et al., 2007); ninein re-localization also occurs during the differentiation of stratified epithelial cells in the mouse epidermis, where it targets to desmosomal junctions (Lechler and Fuchs, 2007). Inhibition of the core desmosomal component, desmoplakin, disrupts ninein targeting and formation of the peripheral non-centrosomal microtubule array (Lechler and Fuchs, 2007), but direct evidence that ninein is important for array formation is currently lacking.

The Patronin/CAMSAP/Nezha family of minus end-associated proteins, conserved among animals with differentiated tissues (Baines et al., 2009), are also implicated in the formation of non-centrosomal arrays (Akhmanova and Hoogenraad, 2015). Members of this protein family are thought to be involved in protecting microtubule minus ends from depolymerizing kinesins (Goodwin and Vale, 2010; Hendershott and Vale, 2014; Jiang et al., 2014). Drosophila and C. elegans each have one family member (Patronin and PTRN-1, respectively), whereas vertebrates have three (calmodulin-regulated spectrin-associated protein or CAMSAP1-3). Although initially identified in cultured epithelial cells (Meng et al., 2008; Jiang et al., 2014), the main in vivo phenotypes associated with knockdown of Patronin/CAMSAP/Nezha family members have been in neurons (Chuang et al., 2014; King et al., 2014; Marcette et al., 2014; Richardson et al., 2014; Yau et al., 2014).

As outlined above, γ-tubulin and Patronin respectively harbor minus-end nucleation and protection activities, and ninein is proposed to anchor microtubules. Mechanistic work has also raised the possibility of functional redundancies between minus end-associated factors. For example, in addition to being a microtubule nucleator, γ-tubulin complexes can cap microtubule minus ends (Keating and Borisy, 2000; Wiese and Zheng, 2000). Similarly, CAMSAP-tubulin stretches may function as seeds that allow microtubule regrowth (Tanaka et al., 2012; Jiang et al., 2014), and both ninein and Patronin family members localize to junctional complexes (Lechler and Fuchs, 2007; Meng et al., 2008) where they could serve anchoring functions. Hence, another important open question is the extent to which minus end-associated factors function collaboratively or redundantly during microtubule array assembly in vivo.

Here, we characterize the C. elegans protein NOCA-1 (non-centrosomal array 1), a protein we identified in a prior high-content screen because its inhibition phenocopied the effect of γ-tubulin removal on germline morphology (Green et al., 2011). We show that NOCA-1 shares homology with vertebrate ninein and identify isoforms that are necessary and sufficient for NOCA-1 function in three different tissues. We explore the functional relationship between NOCA-1, γ-tubulin, and Patronin/PTRN-1 in the assembly of non-centrosomal microtubule arrays. In the larval epidermis, NOCA-1 functions with γ-tubulin in parallel to Patronin/PTRN-1 to assemble a circumferential microtubule array required for larval development. In the germline and embryonic epidermis, NOCA-1 functions independently of Patronin to promote assembly of microtubule arrays required for nuclear positioning. Cumulatively, our results suggest that NOCA-1 functions together with γ-tubulin to direct the assembly of non-centrosomal arrays in multiple tissues and highlight functional overlap between the ninein and Patronin families of microtubule cytoskeleton-controlling proteins.

The piece can be read at eLifeSciences  DOI ( Published September 15, 2015 Cite as eLife 2015;4:e08649 )

Posted on : October 22/10/15

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The
Earth

 

  The
Moon

 

The Lake Eden Eye

 

 

 

 

The Window of the Heavens Always Open and Calling: All We Have to Do Is: To Choose to Be Open, Listen and Respond

 

 

 

Imagine a Rose-Boat

Imagine a rose floating like a tiny little boat on this ocean of infinity
And raise your soul-sail on this wee-little boat and go seeking out
All along feed on nothing but the light that you gather only light
Fear shall never fathom you nor greed can tempt nor illusion divert
For Love you are by name by deeds you are love's working-map

 

 

Only in the transparent pool of knowledge, chiselled out by the sharp incision of wisdom, is seen the true face of what truth is: That what  beauty paints, that what music sings, that what love makes into a magic. And it is life: a momentary magnificence, a-bloom like a bubble's miniscule exposition, against the spread of this awe-inspiring composition of the the Universe. Only through the path of seeking, learning, asking and developing, only through the vehicles and vesicles of knowledge, only through listening to the endless springs flowing beneath, outside, around and beyond our reach, of wisdom, we find the infinite ocean of love which is boundless, eternal, and being infinite, it makes us, shapes us and frees us onto the miracle of infinite liberty: without border, limitation or end. There is nothing better, larger or deeper that humanity can ever be than to simply be and do love. The Humanion

 

Poets' Letter Magazine Archive Poetry Pearl

About The Humanion The Humanion Team Home Contact Submission Guidelines
The Humanion Online Daily from the United Kingdom for the World: To Inspire Souls to Seek

At Home in the Universe : One Without Frontier. Editor: Munayem Mayenin

All copyrights @ The Humanion: London: England: United Kingdom: Contact Address: editor at thehumanion dot com

First Published: September 24: 2015