The structure of epidermal feet during their development

Epidermal cells in Calpodes and other insects form basal processes or feet that at first extend axially and later shorten at the same time as the larval segment shortens to the pupal shape. The feet grow into spaces at the surfaces of other cells to make a basal interlacing meshwork of cellular extensions that are combined mechanically by their desmosomal attachments to cell bodies above and to the basal lamina below. Microtubules and microfilaments are linked to these junctions by a reticular fibrous matrix. Gap junctions on the feet may couple cells that are several cell bodies removed from one another. The meshwork is also a sieve separating the hemolymph from the spaces between cells to form an intercellular compartment. Entry to the intercellular compartment is through the sieve made by the negatively charged basolateral cell surfaces that can prevent the entry of positively charged molecules such as cationic ferritin. As the cells become columnar, coincident with the metamorphic change in segment shape, the feet shorten and pack more densely together. At this time the basal lamina buckles axially as if responding to contraction of the feet. Segment shape change involves cell rearrangement and relative cell movement, necessitating the transient loss of plasma membrane plaque attachments to the cuticle apically and the loss of junctions laterally. Gap junctions involute in characteristic vacuoles. The metamorphic reduction in cell surface area coincides with the loss of basolateral membrane in smooth tubes and vesicles and the turnover of the apical surface in multivesicular bodies. New apical plasma membrane plaques and new lateral and basal junctions stabilize the cells in their pupal positions.     

Haemolymph ecdysteroid titre and epidermal cell commitment of the female southwestern corn borer larvae

The epidermal cell commitment (to pupation or formation of immaculate larvae) and related haemolymph ecdysteroid titres of the southwestern corn borer, Diatraea grandiosella were studied in both nondiapause-bound and diapause-bound last-instar female larvae. Cell commitment was estimated by examining the characteristics of new cuticle secreted in response to an injection of 20-hydroxyecdysone. Haemolymph ecdysteroid titres were determined by radioimmunoassay. Juvenile hormone effect on epidermal cell commitment was studied by applying a juvenile hormone mimic (ZR-515) to last-instar non-diapause-bound larvae and examining the resulting cuticle.In non-diapause-bound larvae, the epidermis of different body regions was committed to pupal development at different times. When pupal cuticular characteristics were evaluated by a scoring system, it appeared that the development of normal pupal cuticle is discontinuous. Three sudden increases in pupal characteristics were observed at 1.67, 2.67 and 3.67 days into the last-larval instar. Haemolymph ecdysteroid titre changes were correlated with the sudden increases in pupal characteristics. Peak ecdysteroid titres were found at 1.67, 2.33, and 3.33 days into the final instar. A fourth ecdysteroid peak (138.8 ng/ml of haemolymph) occurred in pharate pupae. In contrast, the commitment of diapause-bound larvae to produce immaculate integument was made in a fast and continuous fashion. Full commitment was made by 50% of the individuals 4 days (ca. first quarter) into the stadium. Haemolymph ecdysteroid titres fluctuated during the first 2 weeks of the stadium but no significant peaks were observed prior to pharate stage. An ecdysteroid peak (29.8 ng/ml of haemolymph) was identified in pharate immaculate larvae.Pupal development could be completely prevented in 26.7% of nondiapause-bound larvae as late as 4 days into the last instar by topical application of ZR-515. This indicates that the commitment to pupation as revealed by 20-hydroxyecdysone injection is reversible.     

The formation of plasma membrane reticular systems in the oenocytes of an insect

Plasma membrane reticular systems (RSs) are infolds of the plasma membrane found in cells of several insect tissues that are not transporting epithelia. They form a subsurface reticular lymph space that may be involved in the loading and unloading of hemolymph carrier molecules. The development of a new RS during the fifth larval stadium has been studied in the oenocytes of Calpodes ethlius by scanning electron microscopy. The RS forms by the extension and progressive apical fusion of cell processes leaving a reticular lymph space below. Reticular system formation occurs in a front moving over the cell surface. The RS made in the 4th stadium persists through the moult to the 5th stage but diminishes for the next 3 days. A new intermoult RS then forms very quickly. Its time of formation follows the commitment ecdysteroid peak rather than the beginning of secretion by the wax glands. This new 5th stage RS is maintained during the period of intermoult synthesis, after which it declines and is nearly absent by the time of pupation.     

Haemolymph ecdysteroid levels and cellular events in the intermoult/moult sequence of Calpodes ethlius

The haemolymph ecdysteroid titre of the last larval and pupal stadia of Calpodes ethlius was determined by radioimmunoassay. During the last larval stadium, four significant ecdysteroid peaks are present, two of which have been reported for other Lepidoptera. The first peak occurs 12 hr after ecdysis and correlates temporally with nucleolar activity, RNA synthesis and organelle formation in the fat body and epidermis. It correlates also with fat body DNA synthesis, polyploidy and the initiation of a low rate of lipid synthesis. Another peak, at 78 hr, starts its increase when the prothoracic glands no longer require the influence of the brain to produce ecdysone for pupation, and marks the first critical period. It correlates with the initiation of epidermal DNA synthesis and mitosis, and with the progressive determination of pupal characteristics (change in commitment, reprogramming). This ecdysteroid peak may also be involved in the massive intermoult syntheses in the epidermis (lamellate cuticle, wax) and the fat body (lipid, protein). The largest ecdysteroid peak is seen at 162 hr, 6 hr after the tissues no longer require the prothoracic glands for pupation (second critical period). It correlates temporally with the cessation of massive synthetic activity in both epidermis and fat body and initiates preparation for pupal synthesis in both tissues. At this time the ratio of ecdysone: 20-hydroxyecdysone is ～ 1 : 6.6.In common with other Lepidoptera, a single large ecdysteroid peak occurs during the first half of the pupal stadium. Comparisons between these events and the ecdysteroid titre are made between Calpodes and other insects.     

The development of the plasma membrane reticular system in the fat body of an insect

Several insect tissues have plasma membranes that are folded inwards to make a subsurface reticulum on faces that are exposed to hemolymph. The infolds have been called plasma membrane reticular systems (RSs) to distinguish them from the somewhat similar structures found in transporting epithelia. They are characterized by having negative charges on the plasma membranes of the entranceways and by the concentration of some hemolymph proteins in their lymph spaces. Their formation and loss in the fat body has been studied by scanning electron microscopy during the fifth stadium of Calpodes ethlius (Lepidoptera, Hesperiidae). Fat body cells begin the fifth stadium arranged in ribbons with the cells linked together by a fringe of processes. In the first stage many more processes form. These partially fuse together in the second stage, leaving a subsurface reticulum connected by narrow entrances to the lateral cell faces and the face below the basal lamina. Both the cell processes and the reticular systems that they enclose are usually axially orientated. The completed RS persists for the second half of the intermoult devoted to larval syntheses when the concentration of hemolymph proteins rises. After protein sequestration prior to pupation the RS is lost and the fat body returns to being a tissue of rounded cells linked by a few enmeshed processes.     

Physiological and endocrine changes associated with polydnavirus/venom in the parasitoid-host system Chelonus inanitus-Spodoptera littoralis

As shown earlier, parasitization by the egg-larval parasitoid C. inanitus causes in its host the precocious onset of metamorphosis in the 5th instar followed by developmental arrest in the prepupal stage. Polydnavirus/venom were shown to be responsible for the developmental arrest. We investigated how polydnavirus/venom affect growth of the host larvae and found that head capsule widths were smaller from the 4th to 6th stadium and weights were lower in the 6th stadium in polydnavirus/venom-containing larvae than in non-parasitized larvae. In an attempt to identify endocrine parameters that are modified by polydnavirus/venom and might be responsible for the developmental arrest in the prepupa, we compared juvenile hormones, juvenile hormone esterase and ecdysteroids between non-parasitized and polydnavirus/venom-containing larvae from the 4th instar until pupation or developmental arrest, respectively. Obvious differences became manifest only in the 6th instar at the pupal cell formation stage, i.e. 12 days after entry of polydnavirus/venom into the host egg. Then, prothoracic glands of polydnavirus/venom-containing larvae released less ecdysteroids and ecdysteroid titres were lower than in non-parasitized larvae; this was followed by a delayed, reduced and desynchronized increase in prepupal juvenile hormones and juvenile hormone esterase and a slightly modified metabolism of ecdysone. This indicates that polydnavirus/venom affects the endocrine system of the host only after pupal commitment and that inhibition of prothoracic gland activity is the first detectable effect.     

Epidermal feet in pupal segment morphogenesis

Epidermal cells in insect integumental epithelia develop branched cytoskeletal extensions or feet at their base that are similar in appearance to the processes put out by cells in tissue culture. We have developed a procedure to show the feet that gives an effect as if thousands of cells randomly arranged in the epithelium had each been injected with a lead salt visualized as black lead sulphide. The procedure depends upon the fact that after brief glutaraldehyde fixation, tannic acid only penetrates some cells where it mordants lead ions and binds osmium. Individual cells visualized in this manner show their outlines as if they are separate in a tissue culture although they are part of a closely packed epithelium. The feet are metamorphic structures formed after pupal commitment and are necessary for metamorphic changes in segment shape. In Calpodes larvae the feet are orientated axially in the direction of the segmentally repeating gradient and may extend for several cell diameters. They extend under the influence of low titres of 20-hydroxyecdysone such as those occurring in the intermoult. When stimulated by high titres like those in pre-pupae, the feet contract at the same time as the segments shorten to pupal proportions. We believe that cell processes like the epidermal feet are ubiquitous but that they have often been overlooked because of the difficulty of demonstrating the outlines of single cells that are united in epithelia.     

Is cell sorting caused by differences in the work of intercellular adhesion? A critique of the steinberg hypothesis

The differential adhesion hypothesis, developed by Malcolm Steinberg, proposes that the histotypic sorting out behavior of aggregated cells is mechanistically equivalent to certain aspects of liquid surface tension, specifically the spontaneous separation of immiscible liquids of differing surface tension. According to Steinberg's hypothesis, the adhesive forces between aggregated cells play essentially the same role in cell sorting as are played by intermolecular attractive forces in liquid surface tension.In this paper I discuss a number of crucial distinctions between intermolecular attraction (in liquids) and intercellular adhesion (in aggregates). First, liquid drops are closed systems thermodynamically whereas aggregates of living cells can generate an indeterminate amount of metabolic energy capable of altering cell positions and adhesions. Secondly, intercellular adhesions are more than just close range attractions since cells can be held together by forces in addition to those which originally pulled them together. Third, the breakage of intercellular adhesions need not be simply the reverse, thermodynamically, of the formation of those adhesions. And fourthly, because intercellular adhesion is generally concentrated at relatively small foci such as desmosomes, a maximization of intercellular adhesion does not necessarily require a maximization of intercellular contact area, or vice versa.In addition, several alternative hypotheses are proposed, each of which is theoretically capable of explaining cell sorting and the other surface tension-like aspects of cell aggregate behavior which Steinberg has sought to explain as consequences of differential adhesion. In particular, a differential surface contraction hypothesis is proposed, according to which cell sorting and related phenomena are the results of cell surface contractions induced to occur over those portions of the cell surface which are exposed to the surrounding culture medium. Because of the evidence that various invagination type movements of embryonic epithelia are caused by cell surface contractions, it is suggested that differential surface contraction is the most likely explanation of histotypic cell sorting. A number of experiments are suggested by which these various hypotheses might be tested.     

Nucleolar cycles during the fifth stadium in Manduca epidermis

The changing pattern of nucleolar structure in the epidermal cells of Manduca sexta has been correlated with hormonal changes taking place during the fifth stadium. The epidermal nucleoli show three cycles of development, the first and third of which occur at the beginnings of the intermoult and moult phases respectively and are related to larval and pupal syntheses. The second phase occurs in the middle of the stadium but prior to the onset of wandering and commitment to pupation. A phase of mitosis separates the second and third cycles. The three cycles thus correspond in time to those found in Calpodes. The three cycles of nucleolar change are superimposed over nuclear changes relating to the degree of ploidy. Each phase begins with an expansion of the condensed nucleoli to form lobed rings and then necklaces. In the first phase (day 0-3), the rings and necklaces progress to form threaded networks. Both rings and networks have many ribosomal precursor granules that are lacking in condensed nucleoli. The rings and networks are therefore presumed to be more active in rRNA synthesis than the condensed state. The first and third phases of nucleolar change occur after elevated titres of haemolymph ecdysteroid. Post-thoracic ligation of animals at ecdysis blocks nucleolar changes as well as the appearance of polyploid nuclei. Nucleolar changes may be a primary response of the epidermis to stimulation by ecdysone.     

Metamorphic changes in the central projects of hair sensilla inTenebrio molitor L. (Insecta: Coleoptera)

Summary This paper describes the afferent projections of hair sensilla of the pro- and mesothoracic legs and the lateral thoracic sclerites of larval and adultTenebrio molitor and the corresponding set of pupal hair sensilla. The sensory neurons that innervate the hair sensilla of larval or adult insects project somatotopically into the thoracic neuropil. Different types of sensilla on the same region of the body surface project to the same zone of the ipsilateral thoracic ventral neuropil but exhibit different arborization patterns. Although there is a profound reorganization of body surface sensilla, the basic somatotopic layout of the larva is maintained in the adult. The sensory neurons that innervate the pupal hair sensilla possess central projections similar to those of the corresponding adult sensory neurons. The central projections of pupal sensory neurons are somatotopically oriented. Their projection pattern is serially homologous in the thoracic and the abdominal ganglia. The central projection pattern of the described pupal sensory neurons is constant throughout pupation. MAb 22C10 immunoreactivity allows an estimate of the timing of the early differentiation of the imaginal sensory neurons originating during pupation. Ablation experiments indicate that pupal sensory neurons influence the central projection pattern of the differentiating imaginal sensory neurons.     

The involvement of adherens junction components in myofibrillogenesis in cultured cardiac myocytes.

The distribution of adherens junction (AJ) components was investigated in cultured heart myocytes. These cells, derived from either newborn rats or chick embryos, develop elaborate arrays of myofibrils which become extensive and laterally aligned following several days in culture. The Z-disks in these cells, visualized by immunolabeling with antibodies to muscle-specific alpha-actinin, exhibit a characteristic periodicity of about 2 microns and are in register with those of neighboring myofibrils throughout the sarcoplasm. Vinculin, in these cells, associates with intercellular AJ and cell-matrix adhesions. In addition, this protein is detected in periodic bands located along the lateral cell membranes corresponding to "costamers" previously described by Pardo, J.V., Siliciano, J.D. and Craig, S.W. (Proc. Natn. Acad. Sci. USA, 80, 1008). Similarly, N-cadherin, which is predominantly associated with intercellular junctions, is also detected in periodic striations located mainly on the dorsal and lateral cell surfaces. Using computer-aided three-dimensional microscopy confirmed that these vinculin- and N-cadherin-containing structures are located in extrajunctional sites, apparently associated with Z-disks of peripheral myofibrils. Based on these findings an alternative pathway is proposed for the assembly of vinculin and N-cadherin, which is not triggered by adhesive interactions with extracellular surfaces but rather by interactions at the membrane-cytoplasm interphase with the periphery of the pre-assembled myofibrils. Moreover, we present evidence that antibodies to N-cadherin, which are capable of blocking AJ formation in culture, have an inhibitory effect also on the development and alignment of myofibrils. We discuss the functional significance of the "costameric" organization of vinculin and N-cadherin and consider its involvement both in the lateral alignment of neighboring muscle cells and in the stabilization of developing myofibrils.     

An ultrastructural study of cell junctions and the cytoskeleton in epithelial cells of the molluscan integument

Cell junctions and the cytoskeleton of integumental epidermal cells from six bivalves, four gastropods, and two cephalopods were studied by transmission electron microscopy. In all species examined, the junctions in supporting cells presented the following similar pattern: an apical-lateral adhesion belt (occluding junctions were not observed); (b) a lateral complex of septate junctions and smooth septate junctions, with interdigitations between adjacent cells while the gap junctions were not constantly present, and a basal complex with hemidesmosomes, focal contacts, and sometimes basolateral adherent junctions. Desmosomes were never observed. Microfilamentous and microgranular material were present throughout the cells, as bundles of microfilaments within microvilli and the terminal web, within interdigitations, and as cytoplasmic plaques forming part of the adherent junctions, hemidesmosomes, and focal contacts. Bundles of intermediate filaments that originated from basal hemidesmosomes were located close to and oriented parallel with the lateral plasma membrane and terminated within the terminal web. In cells of Aplysia depilans, intermediate filaments converged apically to terminate in hemidesmosome-like structures at the bases of the microvilli. In the cephalopods, hemidesmosomes were never observed and intermediate filaments made direct contact with the basal cell membrane. Some functional interpretations and hypotheses were also discussed.     

Surface architecture of the mucosal epithelium of the cat trachea: I. Cartilaginous portion

The mucosal covering of the cartilaginous portion of the cat trachea was studied by correlated light, transmission-electron, and scanning-electron microscopy. While in some areas the ciliated pseudostratified epithelial lining is fairly smooth in contour, in other areas it contains longitudinally oriented, cilia-lined clefts. Ducts from submucosal glands sometimes open into the base of these clefts, or into funnel-shaped stomata that are lined by either ciliated or microvillus-rich cells. Specialized epithelial cells are occasionally associated with the clefts or with other regions of surface epithelium. In single sections, these cells appear to contain a cilium-lined vacuole, but serial sectioning has demonstrated that these apparent vacuoles actually are long intracellular invaginations in enormously elongated cells that extend longitudinally in the plane of the epithelium. The function of these cells is undetermined. Basal cells are attached to the lamina densa by means of hemidesmosomes that consistently lack peripheral densities; in contrast, the tall columnar cells have no hemidesmosomes.     

Ultrastructure and morphology of midgut visceral muscle in early pupal Aedes aegypti mosquitoes

These studies focus on the pupal Aedes aegypti midgut muscularis for the first 26 h following larval-pupal transition. The midgut muscularis of Ae. aegypti pupae during this first half of the pupal stadium is a grid of both circularly and longitudinally oriented muscle bands, arranged in a manner resembling that of the larvae. While many muscle bands exhibit signs of degeneration during the time period studied, not all bands degrade, nor is this degradation simultaneous. Band deterioration involves destruction of internal elements while the muscle fiber plasma membrane remains intact. Deterioration of contractile elements may involve proteosome-like structures and associated enzymes. Many features of the larval muscularis including cruciform cells, bifurcating circular bands, and bifurcating longitudinal bands of muscle are retained during the time period investigated. Neuromuscular junctions along some muscle bands are retained through at least 16 h into the pupal stadium. The selective nature of muscle fiber degradation, coupled with the retention of larval features and neural input, may allow for limited functionality of the muscularis during metamorphosis. Evidence of sexual dimorphism in the midgut muscularis of male and female Ae. aegypti pupae was not observed during the time period studied.     

Cell junctions in amphioxus (Cephalochordata): a thin section and freeze-fracture study

Tissues from the epidermis, alimentary tract and notochord of the cephalochordate Branchiostoma lanceolatum have been examined in both thin sections and freeze-fracture replicas to ascertain the nature of the intercellular junctions that characterize their cell borders. The columnar epithelial cells from the branchial chamber (pharynx), as well as from the anterior and posterior intestine, all feature cilia and microvilli on their luminal surfaces. However, their lateral surfaces exhibit zonulae adhaerentes only. No gap junctions have been observed, nor any tight junctions (as are a feature of the gut of urochordates and higher vertebrates), nor unequivocal septate junctions (as are typical of the gut of invertebrates). The basal intercellular borders are likewise held together by zonulae adhaerentes while hemidesmosomes occur along the basal surface where the cells abut against the basal lamina. The lateral cell surfaces, where the adhesive junctions occur, at both luminal and basal borders, do not exhibit any specialized arrangement of intramembrane particles (IMPs), as visualized by freeze-fracture. The IMPs are scattered at random over the cell membranes, being particularly prevalent on the P-face. The only distinctive IMPs arrays are those found on the ciliary shafts in the form of ciliary necklaces and IMP clusters. With regard to these ciliary modifications, cephalochordates closely resemble the cells of the branchial tract of ascidians (urochordates). However, the absence of distinct junctions other than zonulae adhaerentes makes them exceptions to the situation generally encountered in both vertebrates and urochordates, as well as in the invertebrates. Infiltration with tracers such as lanthanum corroborates this finding; the lanthanum fills the extracellular spaces between the cells of the intestine since there are no junctions present to restrict its entry or to act even as a partial barrier. Junctions are likewise absent from the membranes of the notochord; the membranes of its lamellae and vesicles exhibit irregular clusters of IMPs which may be related to the association between the membranes and the notochordal filaments. Epidermis and glial cells from the nervous system possess extensive desmosomal-like associations or zonulae adhaerentes, but no other junctional type is obvious in thin sections, apart from very occasional cross-striations deemed by some previous investigators to represent 'poorly developed' septate junctions.     

Cell Junctions in Arthropod Ion-transport Systems

The epithelial cells involved in the movement of ions and waterform a major subset of all epithelial cell types. Both the formand the functions of cell junctions present in these cells areessentially the same as those found elsewhere. Gap junctionsare believed to regulate intercellular communication; desmosomesand hemidesmosomes provide mechanical anchorage to other cellsand the extracellular matrix; septate junctions play roles inproviding cell to cell anchorage, and perhaps in sealing thelateral surfaces of adjacent cells together to prevent paracellularfluid and solute movement; tight junctions (of limited distributionin insects) are seals between adjacent cells. They form a barrierto the paracellular movement of solutes and water. Examination of the junctions in salivary glands and midgut provideinsight into the roles of these junctions in the developmentand function of ion transport systems. In Manduca sexta (Johannsen)the cells of the salivary gland are joined by pleated septateand gap junctions. Individual salivary cells have numerous foldsand canaliculi. The walls of the canaliculi consist of extensivelyfolded plasma membrane in intimate association with mitochondria.Gap junctions connect adjacent parts of the same cell acrossmembrane folds, effectively shortening diffusion distances inthe cells. Hemidesmosomes are present in the walls of developingcanaliculi. They are attached to pore filaments that occupythe lumen of the developing canaliculi. The hemidesmosomes andpore filaments may have a morphogenetic role as they disappearafter the canaliculi are formed. In Manduca sexta the midgut cells are joined by gap and septatejunctions. These junctions differ in morphology from their counterpartsin the salivary gland; physiological studies show the gobletcells are not coupled to neighboring tall columnar cells. Wehave shown the gap junctions joining them are typical of non-couplingjunctions. Preliminary studies suggest that the gap junctionschange form when the cells are coupled.     

CELLULAR DIFFERENTIATION IN THE URINARY BLADDER OF A EURYHALINE MARINE FISH, GILLICHTHYS MIRABILIS, IN RESPONSE TO ENVIRONMENTAL SALINITY CHANGE

The urinary bladder of a euryhaline marine teleost, Gillichthys mirabilis , was studied by light and electron microscopy. An enlargement of the mesonephric ducts forms a sac-like structure lined by an epithelium composed of two major cell types. Tall columnar cells continuous with the duct epithelium are characterized by a large number of mitochondria and well-developed rough and smooth endoplasmic reticulum. Tubular smooth endoplasmic reticulum is more developed in the basal cytoplasm and often opens into the extracellular space. A second cell type, the low cuboidal cells, forming most of the bladder epithelium, has fewer mitochondria. Basal cells are rarely observed and mucous cells are absent.
In seawater Gillichthys , cells of both types are separated by narrow intercellular spaces. In 5% seawater fish, the columnar cells show functional activation, as evidenced by an increased number of mitochondria and more extensive tubular smooth endoplasmic reticulum. No such changes were noted in the cuboidal cells; however, the lateral intercellular spaces are dilated probably owing to hypotonicity of the urine in the hypotonic environment. A functional difference between the two cell types is strongly suggested. The columnar cells may be responsible for active sodium uptake in hypotonic seawater environments.     

Separation of distinct adhesion complexes and associated cytoskeleton by a micro-stencil-printing method

Adhesion between cells and the extracellular matrix is mediated by different types of transmembraneous proteins. Their associations to specific partners lead to the assembly of contacts such as focal adhesions and hemidesmosomes. The spatial overlap between both contacts within cells has however limited the study of each type of contact. Here we show that with “stampcils” focal contacts and hemidesmosomes can be spatially separated: cells are plated within the cavities of a stencil and the grids of the stencil serve as stamps for grafting an extracellular matrix protein—fibronectin. Cells engage new contacts on stamped zones leading to the segregation of adhesions and their associated cytoskeletons, i.e., actin and intermediate filaments of keratins. This new method should provide new insights into cell contacts compositions and dynamics.     

Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing

Cell elongation and polarization are basic morphogenetic responses to extracellular matrix adhesion. We demonstrate here that human cultured fibroblasts readily polarize when plated on rigid, but not on compliant, substrates. On rigid surfaces, large and uniformly oriented focal adhesions are formed, whereas cells plated on compliant substrates form numerous small and radially oriented adhesions. Live-cell monitoring showed that focal adhesion alignment precedes the overall elongation of the cell, indicating that focal adhesion orientation may direct cell polarization. siRNA-mediated knockdown of 85 human protein tyrosine kinases (PTKs) induced distinct alterations in the cell polarization response, as well as diverse changes in cell traction force generation and focal adhesion formation. Remarkably, changes in rigidity-dependent traction force development, or focal adhesion mechanosensing, were consistently accompanied by abnormalities in the cell polarization response. We propose that the different stages of cell polarization are regulated by multiple, PTK-dependent molecular checkpoints that jointly control cell contractility and focal-adhesion-mediated mechanosensing.