The highly elongated Drosophila mechanosensory bristle - a new model for studying polarized microtubule function

Microtubules (MTs) are polar polymers that can facilitate asymmetric distribution of cell components, a process important for polarized cell growth. The highly elongated and polarized Drosophila mechnosensory bristle cytoplasm is filled with short MTs that constitute a significant component of the shaft cytoplasm. Inhibition of MT assembly affects biased axial growth in the bristle and highlights the importance of MTs for this process. We demonstrate that the vast majority of bristle MTs are organized in a polarized manner, minus-ends out. We also show that genetic disruption of the MT polarity affects the polar distribution of cell components and leads to an alteration in the biased axial shape of the bristle shaft. Thus, we suggest that the asymmetric organization of the MT population within the bristle cell shaft is necessary for the proper axial elongation of this cellular extension. We would also like to emphasize the benefits of using the bristle cell as a model for studying MTs and MT-binding proteins because changes to this cytoskeletal component result in easily recognized at the phenotypes.     

Asymmetric Microtubule Function Is an Essential Requirement for Polarized Organization of the Drosophila Bristle

While previous studies have shown that microtubules (MTs) are essential for maintaining the highly biased axial growth of the Drosophila bristle, the mechanism for this process has remained vague. We report that the MT minus-end marker, Nod-KHC, accumulates at the bristle tip, suggesting that the MT network in the bristle is organized minus end out. Potential markers for studying the importance of properly polarized MTs to bristle axial growth are Ik2 and Spindle-F (Spn-F), since mutations in spn-F and ik2 affect bristle development. We demonstrate that Spn-F and Ik2 are localized to the bristle tip and that mutations in ik2 and spn-F affect bristle MT and actin organization. Specifically, mutation in ik2 affects polarized bristle MT function. It was previously found that the hook mutant exhibited defects in bristle polarity and that hook is involved in endocytic trafficking. We found that Hook is localized at the bristle tip and that this localization is affected in ik2 mutants, suggesting that the contribution of MTs within the bristle shaft is important for correct endocytic trafficking. Thus, our results show that MTs are organized in a polarized manner within the highly elongated bristle and that this organization is essential for biased bristle axial growth.Polarized cell growth, manifested as cellular growth biased toward one pole of a cell, is the result of dynamic developmental processes that require an extensive reorganization of the cytoplasm in response to both intracellular and extracellular signals. Essentially, all cells can polarize in response to internal and/or external cues, such as matrix components, cell-cell contacts, or chemical gradients. Eukaryotic cells generally interpret these cues by assembling a polarized actin cytoskeleton at the cortex, which in turn coordinates with microtubules to guide internal membranes. This network ultimately polarizes events that occur internally and at the cell surface (10). A critical issue in this respect concerns how the cytoskeleton responds to those cues that lead to polarized growth.During development, Drosophila epidermal cells form a variety of polarized structures. These include the epidermal hairs that decorate much of the adult cuticular surface, the shafts of the bristle sense organs, the lateral extensions of the arista, and the larval denticles. These cuticular structures are produced by cytoskeleton-mediated outgrowths of the epiderma (13, 16). Since alterations in bristle morphology are easy to detect in living flies and since small changes in the actin cytoskeleton, as induced by drugs or mutations, often result in an easily detectable phenotype, the growth of the bristle cell is used to define the role of the cytoskeleton in polarized cell growth.Bristle cells sprout during metamorphosis and elongate over the course of ∼18 h. Growth is driven by actin filament polymerization (41). The actin bundles in bristle sprouts begin as microvilli (45) and are cross-bridged into modular bundles 1 to 5 μm in length by at least two cross-linking proteins, forked and fascin (43, 45, 46). These modules are then grafted together by end-to-end joining into stiff bundles (15) which run longitudinally along the bristle shaft, attached to the plasma membrane (40), to support the cell extension as well-spaced ribs. Bundles are tapered, with the largest cross-sectional area of individual bundles found at the base, containing >500 filaments (40). In Drosophila pupae, developing bristles contain 7 to 11 (microchaeta) or 12 to 18 (macrochaeta) bundles of cross-linked actin filaments and a large population of microtubules (MTs) that run longitudinally along the bristle shaft. It was suggested that bristle MTs are highly stable, forming at the start of elongation and then moving out along the shaft as the cell elongates (44). Inhibitor studies suggest that MTs are essential for maintaining bristle axial growth, since injection of microtubule antagonists, such as vinblastine, into pupae resulted in short and fat bristles (13).It was previously demonstrated that mutations in the Drosophila ikkɛ homologue, ik2, and in the novel gene spindle-F (spn-F), which is not conserved outside insect species, affect both egg chamber polarity and bristle development (1, 37). During oogenesis, both ik2 and spn-F affect mRNA localization due to their effects on actin and MT minus-end organization. Moreover, we were able to show that Ik2 and Spn-F form a complex that regulates cytoskeleton organization during Drosophila oogenesis, with Spn-F serving as the direct regulatory target for Ik2 kinase activity (11). Further evidence for the role of ik2 in cytoskeleton-related processes comes from its interaction with the Drosophila inhibitor of apoptosis 1 (DIAP1). It was suggested that ik2 acts as a negative regulator of F-actin assembly and maintains the fidelity of polarized elongation during cell morphogenesis by modulating DIAP1 levels (22, 29). Recently it was shown that ik2 regulates the dendrite pruning involved in MT disassembly (23).Since ik2 and spn-F affect bristle polarity organization, we investigated the role of these genes in shaping bristle morphology. We report that MTs within the bristle are organized in a polarized manner, minus-end out. We also demonstrate that both the Spn-F and Ik2 proteins are localized to the bristle tip. Close examination during the bristle elongation period revealed that mutations in either gene affect cytoskeleton organization. Specifically, upon mutation of ik2, the MT minus-end marker is no longer accumulated at the bristle tip. Moreover, we found that the Hook protein is localized at the bristle tip and that such localization is affected in spn-F and ik2 mutants, suggesting that MT functionality within the bristle is essential for recruitment of components of the endocytic trafficking to the tip of the bristle. Thus, we suggest that ik2 and spn-F affect MT functions which are required for the biased axial shape of the bristle. This, in turn, affects the localization of the endocytic trafficking machinery to the bristle tip.     

Dusky-like functions as a Rab11 effector for the deposition of cuticle during Drosophila bristle development

The morphogenesis of Drosophila sensory bristles is dependent on the function of their actin and microtubule cytoskeleton. Actin filaments are important for bristle shape and elongation, while microtubules are thought to mediate protein and membrane trafficking to promote growth. We have identified an essential role for the bristle cuticle in the maintenance of bristle structure and shape at late stages of bristle development. We show that the small GTPase Rab11 mediates the organized deposition of chitin, a major cuticle component in bristles, and disrupting Rab11 function leads to phenotypes that result from bristle collapse rather than a failure to elongate. We further establish that Rab11 is required for the plasma membrane localization of the ZP domain-containing Dusky-like (Dyl) protein and that Dyl is also required for cuticle formation in bristles. Our data argue that Dyl functions as a Rab11 effector for mediating the attachment of the bristle cell membrane to chitin to establish a stable cuticle. Our studies also implicate the exocyst as a Rab11 effector in this process and that Rab11 trafficking along the bristle shaft is mediated by microtubules.     

Actin filament turnover regulated by cross-linking accounts for the size, shape, location, and number of actin bundles in Drosophila bristles

Drosophila bristle cells are shaped during growth by longitudinal bundles of cross-linked actin filaments attached to the plasma membrane. We used confocal and electron microscopy to examine actin bundle structure and found that during bristle elongation, snarls of uncross-linked actin filaments and small internal bundles also form in the shaft cytoplasm only to disappear within 4 min. Thus, formation and later removal of actin filaments are prominent features of growing bristles. These transient snarls and internal bundles can be stabilized by culturing elongating bristles with jasplakinolide, a membrane-permeant inhibitor of actin filament depolymerization, resulting in enormous numbers of internal bundles and uncross-linked filaments. Examination of bundle disassembly in mutant bristles shows that plasma membrane association and cross-bridging adjacent actin filaments together inhibits depolymerization. Thus, highly cross-bridged and membrane-bound actin filaments turn over slowly and persist, whereas poorly cross-linked filaments turnover more rapidly. We argue that the selection of stable bundles relative to poorly cross-bridged filaments can account for the size, shape, number, and location of the longitudinal actin bundles in bristles. As a result, filament turnover plays an important role in regulating cytoskeleton assembly and consequently cell shape.     

The role actin filaments play in providing the characteristic curved form of Drosophila bristles

Drosophila bristles display a precise orientation and curvature. An asymmetric extension of the socket cell overlies the newly emerging bristle rudiment to provide direction for bristle elongation, a process thought to be orchestrated by the nerve dendrite lying between these cells. Scanning electron microscopic analysis of individual bristles showed that curvature is planar and far greater near the bristle base. Correlated with this, as development proceeds the pupa gradually recedes from the inner pupal case (an extracellular layer that encloses the pupa) leading to less bristle curvature along the shaft. We propose that the inner pupal case induces elongating bristles to bend when they contact this barrier. During elongation the actin cytoskeleton locks in this curvature by grafting together the overlapping modules that comprise the long filament bundles. Because the bristle is curved, the actin bundles on the superior side must be longer than those on the inferior side. This is accomplished during grafting by greater elongation of superior side modules. Poor actin cross-bridging in mutant bristles results in altered curvature. Thus, the pattern of bristle curvature is a product of both extrinsic factors-the socket cell and the inner pupal case--and intrinsic factors--actin cytoskeleton assembly.     

The genetic control of arista lateral morphogenesis in Drosophila

The sensory bristles and epidermal hairs of Drosophila have proven to be valuable model cell types for studying the role of the cytoskeleton in cellular morphogenesis. We have recently begun to use the arista laterals as a third model cell type. The laterals display a combination of bristle and hair characteristics and provide a system where we can compare the relative importance of specific genes and subcellular structures for the morphogenesis of different polarized cellular extensions. We have characterized the lateral phenotype of a collection of mutations selected because of their phenotypes in hairs and bristles. In many but not all ways the lateral phenotypes are similar to the hair and bristle phenotypes. We provide compelling genetic evidence for the importance of the actin cytoskeleton in lateral elongation, shaping and integrity. Our observations provide evidence that defects in actin bundling can destabilize laterals so that they split during growth. Temperature shift experiments suggest that a defect in lateral initiation can lead to subsequent splitting. These observations provide a link between multiple hair and lateral cells forming by both multiple initiation events and by the splitting of individual cellular extensions. We also found that mutations that lead to lateral splitting typically alter the stereotypic arrangement of actin filament bundles and microtubules in laterals.     

Nocodazole, vinblastine and taxol at low concentrations affect fibroblast locomotion and saltatory movements of organelles

Microtubules (MTs) are essential for the maintenance of asymmetric cell shape and motility of fibroblasts. MTs are considered to function as rails for organelle transport to the leading edge. We investigated the relationship between the motility of Vero fibroblasts and saltatory movements of particles in their lamella Fibroblasts extended their leading edges into the experimental wound at a rate of 20+/-11 microm/h. Intracellular particles in the front parts of the polarized fibroblasts moved saltatorily mainly along the long axis of the cells. MT depolymerization induced by the nocodazole at a high concentration (1.7 microM) resulted in the inhibition of both fibroblast motility and saltatory movements of the particles. Taxol (1 microM) inhibited the fibroblast locomotion but not the saltatory movements. The saltatory movement pattern was disorganized by taxol by decreasing the portion of longitudinal saltations and consequently by increasing the part of saltations perpendicular to the cell long axis. This effect may be explained by disorganization of the MT network resulting from the inhibition of dynamic instability. To further investigate the relationships between the MT dynamics instability, saltatory movements, and fibroblast locomotion, we treated fibroblasts with microtubule drugs at low concentration (nocodazole, 170 nM; vinblastine, 50 nM; and taxol, 50 nM). All these drugs induced rapid disorganization of the saltatory movements and decreased the rate of cell locomotion. Simultaneously, the amount of acetylated (stable) MTs increased. The treatment also induced reversible changes in the actin meshwork. We suggest that decrease in the fibroblast locomotion rate in the case of MT stabilization occurred because of the appearance of numerous free MTs. Saltations along free MTs are poorly organized and, as a result, the number of organelles reaching the fibroblast leading edge decreases.     

Ultrastructural Observation on Microtubules of the Generative Cell in Amaryllis Germinated Pollens and Pollen Tubes

The organization of microtubules (MTs) in the generative cell (GC) of germinated pollen and pollen tube in Amaryllis vittata Ait. has been studied with electron microscopy. At the beginning of pollen germination, the GC is long elliptic in shape, and is surrounded by its own membrane and also by that of the vegetative cell (VC) ,both of which appear undulated. In cross section, the GC appears roundish and has many lobes. The MT system of GC is mainly organized in bundles, but single MTs can also be observed. The MT bundles are generally located in the lobes, directly beneath the plasma membrane of the cell. These MT bundles orientate along the longitudinal axis of the cell. They are formed by aggregation of 5–6 MTs at least,more often about 30 MTs. In the bundles the MTs are often linked to each other by "cross-bridge". The single tubules in the eytopiasm distribute randomly in different orientations. When the GC has migrated into the pollen tube after germination ,it becomes elongated and has cytoplasmic extensions both in the anterior and posterior end of the cell. The organization of MTs of the GC in pollen tube is similar to that in the germinated pollen grain,but the number of MTs in a bundle often increases to 50–60. In the bundle the "cross-bridges" between the MTs which always link 3–5 MTs, are still seen clearly. Positional shift between the GC and Vegetative nucleus (VN) may take place during the growth of pollen tube. The physical association between GC and VN may be demonstrated some ultrastructural figures. It may be seen that irregular cytoplasmic extensions in the anterior end of the GC is always enclosed by the VN and the projections of the cytoplasmic extensions lie within enclaves of the VN. There are many MTs sheets in the lobes or extensions in the cytoplasm of the GC. Thus the present study demonstrates that MTs have an important role in maintaining the peculiar shape of the GC and the close association between GC and VN. However, it seems that the MTs are probably also engaged in the movement of the GC during pollen growth.     

Dynein-dependent motility of microtubules and nucleation sites supports polarization of the tubulin array in the fungus Ustilago maydis

Microtubules (MTs) are often organized by a nucleus-associated MT organizing center (MTOC). In addition, in neurons and epithelial cells, motor-based transport of assembled MTs determines the polarity of the MT array. Here, we show that MT motility participates in MT organization in the fungus Ustilago maydis. In budding cells, most MTs are nucleated by three to six small and motile gamma-tubulin-containing MTOCs at the boundary of mother and daughter cell, which results in a polarized MT array. In addition, free MTs and MTOCs move rapidly throughout the cytoplasm. Disruption of MTs with benomyl and subsequent washout led to an equal distribution of the MTOC and random formation of highly motile and randomly oriented MTs throughout the cytoplasm. Within 3 min after washout, MTOCs returned to the neck region and the polarized MT array was reestablished. MT motility and polarity of the MT array was lost in dynein mutants, indicating that dynein-based transport of MTs and MTOCs polarizes the MT cytoskeleton. Observation of green fluorescent protein-tagged dynein indicated that this is achieved by off-loading dynein from the plus-ends of motile MTs. We propose that MT organization in U. maydis involves dynein-mediated motility of MTs and nucleation sites.     

F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments

The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper.     

Mutational analysis of Stubble-stubbloid gene structure and function in Drosophila leg and bristle morphogenesis

The Stubble-stubbloid (Sb-sbd) gene is required for ecdysone-regulated epithelial morphogenesis of imaginal tissues during Drosophila metamorphosis. Mutations in Sb-sbd are associated with defects in apical cell shape changes critical for the evagination of the leg imaginal disc and with defects in assembly and extension of parallel actin bundles in growing mechanosensory bristles. The Sb-sbd gene encodes a type II transmembrane serine protease (TTSP). Here we use a Sb-sbd transgenic construct to rescue both bristle and leg morphogenesis defects in Sb-sbd mutations. Molecular characterization of Sb-sbd mutations and rescue experiments with wild-type and modified Sb-sbd transgenic constructs show that the protease domain is required for both leg and bristle functions. Truncated proteins that express the noncatalytic domains without the protease have dominant effects in bristles but not in legs. Leg morphogenesis, but not bristle growth, is sensitive to Sb-sbd overexpression. Antibody localization of the Sb-sbd protein shows apical expression in elongating legs. Sb-sbd protein is found in the base and shaft in budding bristles and then concentrates at the growing tip when bristles are elongating rapidly. We propose a model whereby Sb-sbd helps coordinate proteolytic modification of extracellular matrix attachments with cytoskeletal changes in both legs and bristles.     

The search and prime hypothesis for growth cone turning

In the fields of axonal and dendritic guidance, there is now a significant accumulation of knowledge of how extracellular signaling molecules activate their cognate growth cone receptors. Relatively little is known about the subsequent activation of intracellular signaling pathways and actin reorganization, and very little is known about how microtubules (MTs) reorganize during growth cone turning. I hypothesize that dynamic MTs are required in order to catalyze the polarized actin assembly necessary for growth cone turning, that MTs and actin filaments promote each other's assembly through positive feedback, that MT stability is enhanced further through the formation of membrane-associated MT attachment sites, and that these MT stabilization events subsequently accelerate axonal/dendritic shaft formation.     

Force‐ and length‐dependent catastrophe activities explain interphase microtubule organization in fission yeast

The cytoskeleton is essential for the maintenance of cell morphology in eukaryotes. In fission yeast, for example, polarized growth sites are organized by actin, whereas microtubules (MTs) acting upstream control where growth occurs. Growth is limited to the cell poles when MTs undergo catastrophes there and not elsewhere on the cortex. Here, we report that the modulation of MT dynamics by forces as observed in vitro can quantitatively explain the localization of MT catastrophes in Schizosaccharomyces pombe. However, we found that it is necessary to add length‐dependent catastrophe rates to make the model fully consistent with other previously measured traits of MTs. We explain the measured statistical distribution of MT–cortex contact times and re‐examine the curling behavior of MTs in unbranched straight tea1Δ cells. Importantly, the model demonstrates that MTs together with associated proteins such as depolymerizing kinesins are, in principle, sufficient to mark the cell poles.     

Mechanical Regulation of Neurite Polarization and Growth: A Computational Study

The densely packed microtubule (MT) array found in neuronal cell projections (neurites) serves two fundamental functions simultaneously: it provides a mechanically stable track for molecular motor-based transport and produces forces that drive neurite growth. The local pattern of MT polarity along the neurite shaft has been found to differ between axons and dendrites. In axons, the neurons’ dominating long projections, roughly 90% of the MTs orient with their rapidly growing plus end away from the cell body, whereas in vertebrate dendrites, their orientations are locally mixed. Molecular motors are known to be responsible for cytoskeletal ordering and force generation, but their collective function in the dense MT cytoskeleton of neurites remains elusive. We here hypothesized that both the polarity pattern of MTs along the neurite shaft and the shaft’s global extension are simultaneously driven by molecular motor forces and should thus be regulated by the mechanical load acting on the MT array as a whole. To investigate this, we simulated cylindrical bundles of MTs that are cross-linked and powered by molecular motors by iteratively solving a set of force-balance equations. The bundles were subjected to a fixed load arising from actively generated tension in the actomyosin cortex enveloping the MTs. The magnitude of the load and the level of motor-induced connectivity between the MTs have been varied systematically. With an increasing load and decreasing motor-induced connectivity between MTs, the bundles became wider in cross section and extended more slowly, and the local MT orientational order was reduced. These results reveal two, to our knowledge, novel mechanical factors that may underlie the distinctive development of the MT cytoskeleton in axons and dendrites: the cross-linking level of MTs by motors and the load acting on this cytoskeleton during growth.     

Rho Guanosine Triphosphatase Mediates the Selective Stabilization of Microtubules Induced by Lysophosphatidic Acid

The asymmetric distribution of stable, posttranslationally modified microtubules (MTs) contributes to the polarization of many cell types, yet the factors controlling the formation of these MTs are not known. We have found that lysophosphatidic acid (LPA) is a major serum factor responsible for rapidly generating stable, detyrosinated (Glu) MTs in serum-starved 3T3 cells. Using C3 toxin and val14 rho we showed that rho was both necessary and sufficient for the induction of Glu MTs by LPA and serum. Unlike previously described factors that induce MT stability, rho induced the stabilization of only a subset of the MTs and, in wound-edge cells, these stable MTs were appropriately oriented toward the leading edge of the cell. LPA had little effect on individual parameters of MT dynamics, but did induce long states of pause in a subset of MTs near the edge of the cell. Rho stimulation of MT stability was independent of actin stress fiber formation. These results identify rho as a novel regulator of the MT cytoskeleton that selectively stabilizes MTs during cell polarization by acting as a switch between dynamic and stable states of MTs rather than as a modulator of MT assembly and disassembly.     

The tricornered gene, which is required for the integrity of epidermal cell extensions, encodes the Drosophila nuclear DBF2-related kinase

During their differentiation epidermal cells of Drosophila form a rich variety of polarized structures. These include the epidermal hairs that decorate much of the adult cuticular surface, the shafts of the bristle sense organs, the lateral extensions of the arista, and the larval denticles. These cuticular structures are produced by cytoskeletal-mediated outgrowths of epidermal cells. Mutations in the tricornered gene result in the splitting or branching of all of these structures. Thus, tricornered function appears to be important for maintaining the integrity of the outgrowths. tricornered mutations however do not have major effects on the growth or shape of these cellular extensions. Inhibiting actin polymerization in differentiating cells by cytochalasin D or latrunculin A treatment also induces the splitting of hairs and bristles, suggesting that the actin cytoskeleton might be a target of tricornered. However, the drugs also result in short, fat, and occasionally malformed hairs and bristles. The data suggest that the function of the actin cytoskeleton is important for maintaining the integrity of cellular extensions as well as their growth and shape. Thus, if tricornered causes the splitting of cellular extensions by interacting with the actin cytoskeleton it likely does so in a subtle way. Consistent with this possibility we found that a weak tricornered mutant is hypersensitive to cytochalasin D. We have cloned the tricornered gene and found that it encodes the Drosophila NDR kinase. This is a conserved ser/thr protein kinase found in Caenorhabditis elegans and humans that is related to a number of kinases that have been found to be important in controlling cell structure and proliferation.     

A Highlights from MBoC Selection: Persistent growth of microtubules at low density

Microtubules (MTs) often form a polarized array with minus ends anchored at the centrosome and plus ends extended toward the cell margins. Plus ends display behavior known as dynamic instability—transitions between rapid shortening and slow growth. It is known that dynamic instability is regulated locally to ensure entry of MTs into nascent areas of the cytoplasm, but details of this regulation remain largely unknown. Here, we test an alternative hypothesis for the local regulation of MT behavior. We used microsurgery to isolate a portion of peripheral cytoplasm from MTs growing from the centrosome, creating cytoplasmic areas locally depleted of MTs. We found that in sparsely populated areas MT plus ends persistently grew or paused but never shortened. In contrast, plus ends that entered regions of cytoplasm densely populated with MTs frequently transitioned to shortening. Persistent growth of MTs in sparsely populated areas could not be explained by a local increase in concentration of free tubulin subunits or elevation of Rac1 activity proposed to enhance MT growth at the cell leading edge during locomotion. These observations suggest the existence of a MT density–dependent mechanism regulating MT dynamics that determines dynamic instability of MTs in densely populated areas of the cytoplasm and persistent growth in sparsely populated areas.     

Dynamics and the life cycle of cell microtubules

In living cells microtubules (MTs) continuously grow and shorten. This feature of MTs was discovered in vitro and named dynamic instability. Comparison of dynamic instability of MTs in vitro and in vivo shows a number of differences. MTs in vivo rapidly grow (up to 20 microns/min), duration of their shortening is small (on average 15-20 s), and pauses are prominent. In different animal cells MTs grow from the centrosome and form a radial array. In such cells growth of MTs is persistent, i.e. undergo without interruptions until plus end of a MT reaches cell margin. Analysis of literature and original data shows that interconvertion between phases of growth, shortening and pause is asymmetric: growth often converts into pause, while shortening always converts into growth without pause. We suggest dynamic instability described near the cell margin in numerous publications results not only from intrinsic properties of MTs, but also because of the external obstacles for their growth. MT behavior in the cells with radial array of long MTs could be treated as dynamic instability with boundary conditions. One boundary is the centrosome responsible for rapid initiation of MT growth. Another boundary is cell margin limiting MT elongation. MT growth occurs with constant mean velocity, and potential duration of growth phase might exceed cell radius. MT shortening is usually smaller than MT length however velocity of shortening increases with time. Random episodes of rapid shortening are sufficient for the exchange of MTs in 10-20 min in the cells not more than 40-50 microns in diameter. Experimental data show that similar rate of exchange of MTs is in the large cells. This is achieved employing another mechanism, namely release of MTs and depolymerization from the minus end. In the minus end pathway time required for the exchange of MTs does not depend on cell radius and is determined primarily by the frequency of releases. Thus a small number of free MTs with metastable minus ends significantly reduce time required for the renovation of the radial MT array. Summarizing all experimental data we suggest the life cycle scheme for the MT in a cell. MT is initiated at the centrosome and grows rapidly until it reaches cell margin. At the margin the plus end oscillates, and finally MT depolimerizes. MT "death" comes from a random catastrophe (shortening from the plus end) in small cells or from release and depolymerization of the minus end in large cells.     

Control of bract formation in Drosophila: poxn, kek1, and the EGF-R pathway

In Drosophila, the sensory organs are formed by cells that derive from a precursor cell through a fixed lineage. One exception to this rule is the bract cell that accompanies some of the adult bristles. The bract cell is derived from the surrounding epidermis and is induced by the bristle cells. On the adult tibia, bracts are associated with all mechanosensory bristles, but not with chemosensory bristles. The differences between chemosensory and mechanosensory lineages are controlled by the selector gene pox-neuro (poxn). Here we show that poxn is also involved in suppressing bract formation near the chemosensory bristles. We have identified the gene kek1, described as an inhibitor of the EGF-R signaling pathway, in a screen for poxn downstream genes. We show that kek1 can suppress bract formation and can interfere with other steps of sensory development, including SMC determination and shaft differentiation.     

Cellular mechanisms in the development of the Drosophila arista

Epidermal cells of Drosophila form a variety of polarized structures during their differentiation. These polarized structures include epidermal hairs, the shafts of sensory bristles, larval denticles and the arista laterals. The arista is the terminal segment of the antenna and consists of a central core and a series of lateral extensions. Here we describe the cellular mechanisms involved in the development of the arista and the morphogenesis of the laterals. We found that the development of the arista is a complex process that involves coordinated cell shape changes, elongation of the central core, apoptosis, nuclear migration, the formation of polyploid cells and the outgrowth of the laterals. This developmental program is highly conserved in the development of the arista in the housefly (Musca domestica). Altering arista cell number in Drosophila by stimulating or inhibiting apoptosis results in an altered number of laterals. Interestingly, the increased number of laterals that result from the inhibition of apoptosis in Drosophila results in an arista whose morphology is reminiscent of the Musca arista. Previous experiments have shown that both the actin and microtubule cytoskeletons have important functions in the cellular morphogenesis of hairs and bristles. Inhibitor studies reported here show that this is also the case for the formation of the arista laterals, arguing that the actin and microtubule cytoskeletons have similar functions in the morphogenesis of all of these cell types. We conclude that the arista laterals are a valuable complementary cell type system for studying the morphogenesis of polarized cellular extensions in Drosophila.