Gel'fand Im

Rho-dependent formation of epithelial “leader” cells during wound healing

The motile behavior of epithelial cells located at the edge of a large wound in a monolayer of cultured cells was analyzed. The initial cellular response is alignment of the edge with an accompanying formation of tangential marginal actin bundles within individual cells positioned along the wound edge. Later, coherent out-growths of cell masses occur by the formation of special “leader” cells at the tops of outgrowths and “follower” cells along the sides. Leader cells exhibit profound cytoskeletal reorganization, including disassembly of marginal bundles, the realignment of actin filament bundles, and penetration of microtubules into highly active lamellae. Additionally, cell–cell contacts acquire radial geometry indicative of increased contractile tension. Interestingly, leader cells acquire a cytoskeletal organization and motility typical of fibroblasts. IAR-2 cultures stably transfected with a dominant-negative mutant of RhoA or treated with Rho-kinase inhibitor Y-27632 transformed most edge cells into leader-like cells. Alternatively, transfection of cells with constitutively active RhoA suppressed formation of leaders. Thus, expansion of the epithelial sheet involves functional differentiation into two distinct types of edge cells. The transition between these two patterns is controlled by Rho activity, which in turn controls the dynamic distribution and activity of actin filament bundles, myosin II, and microtubules.

Messages conveyed by spinocerebellar pathways during scratching in the cat. II. Activity of neurons of the ventral spinocerebellar tract

(1) The activity of neurons of the ventral spinocerebellar tract (VSCT) during scratching was studied in thalamic and decapitate cats. The neurons were identified antidromically either by stimulation of the hindlimb area in the anterior lobe of the cerebellum (in thalamic cats) or by stimulation of the contralateral ventrolateral funiculus of the spinal cord (in decapitate cats). The scratch reflex was elicited by stimulation of either the pinna (in thalamic cats) or the cervical spinal cord (in decapitate cats). In most experiments, animals were immobilized and the activity of VSCT neurons was recorded during fictitious scratching. (2) During both actual and fictitious scratching, the discharge of VSCT neurons was rhythmically modulated in relation with the scratch cycle: neurons fired in bursts separated with periods of silence. Phases of activity of different neurons were unevenly distributed over the scratch cycle: most neurons fired within the limits of the flexor phase of the cycle. (3) The firing pattern of VSCT neurons during fictitious scratching was similar to that during actual scratching. Therefore, rhythmical burst firing of VSCT neurons is determined mainly by central mechanisms and not by a rhythmical sensory input. (4) The firing pattern of VSCT neurons in decapitate cats was similar to that in thalamic cats. Therefore, rhythmical burst firing of VSCT neurons is determined mainly by the central spinal mechanism and not by supraspinal motor centers. (5) The VSCT neurons which fired in long bursts during the greater part of the flexor phase were usually activated during the latent period of scratching, while those firing later in the cycle were usually either inhibited or not affected during this period. (6) The antidromic response in most VSCT neurons could be evoked from a large number of points in the hindlimb area of the cerebellar anterior lobe, both in the vermis and in the pars intermedia. Due to such extensive branching of axons, each point of the cortex receives signals from neurons firing in different phases of the cycle. But axons of VSCT neurons firing in long bursts during the greater part of the flexor phase terminate more extensively in the pars intermedia, while axons of neurons firing later in the cycle terminate more extensively in the vermis. (7) The functioning of the VSCT is essentially similar to that of the spino-reticulocerebellar pathway (SRCP). Both pathways convey messages about activity of the central spinal mechanism generating the motor output pattern of scratching, but the VSCT is active mainly in the flexor phase of the scratch cycle and the SRCP in the extensor one. A hypothesis is advanced that these pathways monitor activity of different groups of spinal interneurons.

Formation of bundles of microfilaments during spreading of fibroblasts on the substrate

Abstract Electron microscopy was used to study the sites of formation of bundles of parallel microfilaments in the early stages of spreading of normal mouse embryo fibroblasts on the substrate. Bundles of microfilaments were not found in suspended cells. Contact of the surface of spherical cells with the substrate was not sufficient for the formation of bundles: these bundles were not seen near the under surface of cells that were already attached to the substrate but had not yet developed cytoplasmic outgrowths at their periphery. Peripheral cytoplasmic outgrowths (microspikes and lamellar processes) attached to the substrate were found to be the only sites of localization of the first bundles of microfilaments seen in the spreading cells. It is suggested that surface and/or cytoplasm of the newly-formed peripheral cytoplasmic outgrowth may have some special properties necessary for the initiation of the development of microfilament bundles.

Messages conveyed by spinocerebellar pathways during scratching in the cat. I. Activity of neurons of the lateral reticular nucleus

(1) Signals transmitted to the cerebellum by the spino-reticulocerebellar pathway (SRCP) during scratching were studied. For this purpose, the activity of neurons of the lateral reticular nucleus (LRN), which are the last-order neurons of the SRCP, was recorded during scratching in thalamic cats. Scratching was evoked by stimulation of the pinna. LRN neurons were identified antidromically by stimulation of the hindlimb area in the cerebellar anterior lobe. In most experiments, animals were immobilized with Flaxedil, and stimulation of the pinna resulted in fictitious scratching, i.e., in periodical reciprocal activity of flexor and extensor motoneurons typical of actual scratching. (2) During both actual and fictitious scratching, the discharge frequency of LRN neurons was rhythmically modulated in relation with the scratch cycle. Most LRN neurons fired in short high-frequency bursts of spikes which coincided (completely or partly) with the extensor phase of the cycle. In this respect the SRCP differs from the ventral spinocerebellar tract (VSCT) which is maximally active in the flexor phase of the cycle. (3) The firing pattern of LRN neurons during fictitious scratching was similar to that during actual scratching. Therefore, the rhythmical burst firing of LRN neurons is determined mainly by the central mechanisms and not by the rhythmical sensory input. (4) Rhythmical modulation of LRN neurons disappeared after transection of the ipsilateral lateral funiculus of the spinal cord in which spinoreticular fibers are located. On the other hand, considerable reduction of rhythmical activity in descending brainstem-spinal pathways after contralateral hemisection of the spinal cord did not affect the discharge pattern of LRN neurons. These two facts indicate that the SRCP conveys mainly messages about activity of the central spinal mechanisms, and that influences of supraspinal motor centers.on LRN neurons and on spinoreticular neurons are of minor importance. (5) Axonal terminations of LRN neurons are distributed rather evenly over the hindlimb area in the anterior lobe of the cerebellum. Therefore, messages about the events, which happen within the spinal cord in the vicinity of the extensor phase of the cycle, arrive at every point of the hindlimb area.

Wound healing processes in cell cultures

Abstract Reactions of various types of normal and neoplastic cell cultures to the mechanical removal of a part of the culture were studied. 1. 1. Normal cells actively migrated from the edge of the wound on cell-free glass. Usually this migration was limited: most migrating cells remained within a certain zone near the edge. 2. 2. Cells that synthetized or did not synthetize DNA before the operation migrated into the wound in proportions similar to their proportions in other parts of the culture. 3. 3. Migration of cells into the wound was inhibited by colcemide. 4. 4. Most cells migrating into the wound were induced to begin DNA synthesis after a latent period of about 12–18 h. This activation of DNA synthesis was local: the cells in the other parts of operated cultures were not activated. 5. 5. Migration of neoplastic cells into the wound was very slow as compared with that of the normal cells. This migration was not followed by the activation of DNA synthesis in neoplastic cells.

Contact interactions between epitheliocytes and fibroblasts: Formation of heterotypic cadherin-containing adhesion sites is accompanied by local cytoskeletal reorganization

Contact interactions between different cell types play a number of important roles in development, for example in cell sorting, tissue organization, and ordered migration of cells. The nature of such heterocellular interactions, in contrast to interactions between cells of the same type, remains largely unknown. In this report, we present experimental data examining the dynamics of heterocellular interactions between epitheliocytes and fibroblasts, which express different cadherin cell adhesion molecules and possess different actin cytoskeletal organizations. Our analysis revealed two striking features of heterocellular contact. First, the active free edge of an epitheliocyte reorganizes its actin cytoskeleton after making contact with a fibroblast. Upon contact with the leading edge of a fibroblast, epitheliocytes disassemble their marginal bundle of actin filaments and reassemble actin filaments into a geometric organization more typical of a fibroblast lamella. Second, epitheliocytes and fibroblasts form cell–cell adhesion structures that have an irregular organization and are associated with components of cell adhesion complexes. The structural organization of these adhesions is more closely related to the type of contacts formed between fibroblasts rather than to those between epitheliocytes. Heterotypic epithelio-fibroblastic contacts, like homotypic contacts between fibroblasts, are transient and do not lead to formation of stable contact interactions. We suggest that heterocellular contact interactions in culture may be regarded as models of how tissue systems consisting of epithelia and mesenchyme interact and become organized in vivo.

Effect of colcemid on the spreading of fibroblasts in culture.

Abstract The effect of colcemid upon the spreading of mouse embryo fibroblast-like cells on substrates was studied with the aid of time-lapse microcinematography and scanning electron microscopy. Two types of substrates were used: flat glass and narrow strips of glass surrounded by non-adhesive lipid film; on the latter, spreading and polarization of cells proceeded simultaneously. On glass, colcemid did not prevent transition of cells into a well-attached state; however, the time required for this transition increased considerably as compared with control cultures. Similar effects were caused by two other drugs inhibiting the formation of microtubules: colchicine and vinblastine. The intermediate stages of spreading on flat glass had several abnormal features in the colcemid-containing medium: ( a ) the shape of cytoplasmatic outgrowths formed by the cell was altered and their distribution became less regular; ( b ) partial detachment of the attached parts of the cells was very frequent; ( c ) the spreading of various parts of the cell was not well correlated: the central part of the cell could remain unspread long after the spreading of the peripheral part. Similar effects of colcemid were observed in experiments with cells spreading on the narrow strips of the glass. In addition, colcemid prevented stabilization of the cell surface, i.e., differentiation of the cellular edge into active and stable parts. About two-thirds of the cells attached to the narrow strips of glass were completely detached from the substrate in the course of spreading in colcemid-containing medium. The possible mechanisms of the action of colcemid on spreading are discussed and it is suggested that intracellular structures sensitive to colcemid are essential for the coordination of the reactions in various parts of the cell in the course of spreading.

Mechanisms of polarization of the shape of fibroblasts and epitheliocytes: Separation of the roles of microtubules and Rho-dependent actin-myosin contractility.

Cultured fibroblasts possess a characteristic polarized phenotype manifested by an elongate cell body with an anterior lamella whose cell edge is divided into protrusion-forming and inactive zones. Disruption of the fibroblast microtubule cytoskeleton leads to an increase in Rho-dependent acto-myosin contractile activity and concomitant loss of structural polarity. The functional relationship of myosin-driven contractile activity to loss of fibroblast anterior–posterior polarity is unknown. To dissect the roles of microtubule assembly and of Rho-dependent contractility on structural polarization of cells, polarized fibroblasts and nonpolarized epitheliocytes were treated with the microtubule-depolymerizing drug, nocodazole, and/or the Rho kinase inhibitor, Y-27632. Fibroblasts incubated with Y-27632 increased their degree of polarization by developing a highly elongate cell body with multiple narrow processes extended from the edges of the cell. Treatment of fibroblasts with nocodazole, alone or in combination with Rho kinase inhibitor, produced discoid or polygonal cells having broad, flattened lamellae that did not form long lamellar extensions. Single cultured epitheliocytes of the IAR-2 line do not display anterior–posterior polarization. When treated with Y-27632, the cells acquired a polarized, elongate shape with narrow protrusions and wide lamellas. Nocodazole alone or in combination with Y-27632 did not change the discoid shape of epitheliocytes, however treatment with Y-27632 produced thinning of the lamellar cytoplasm. We conclude that microtubules provide the necessary framework for polarization of fibroblasts and epitheliocytes, whereas Rho-regulated contractility modulates the degree of polarization of fibroblasts and completely inhibits polarization in epitheliocytes.

Origin of signals conveyed by the ventral spino-cerebellar tract and spino-reticulo-cerebellar pathway

Summary(1)The “fictitious” scratch reflex was evoked in decerebrate curarized cats by pinna stimulation. Activity of neurons of the ventral spinocerebellar tract (VSCT) from the L4 and L5 segments of the spinal cord as well as of neurons of the spinoreticulo-cerebellar pathway (SRCP) from the lateral reticular nucleus of the medulla oblongata was recorded. Cooling and destruction of different parts of the lumbo-sacral enlargement of the spinal cord were performed.(2)Cooling of the L5 or L6 segment abolished the rhythmic activity in the greater part of the spinal hindlimb centre but did not affect the generation of rhythmic oscillations in the remaining (rostral) segments of the lumbo-sacral enlargement. Under these conditions, neither the rhythmic activity of VSCT neurons located rostral to the thermode nor that of SRCP neurons changed.(3)A normal rhythmic activity of SRCP neurons also persisted after destruction of grey matter in the L3 and L4 segments. It can be concluded that activity of these neurons is independent of whichever part of the enlargement generates rhythmic oscillations.(4)From these observations a hypothesis is advanced that the main content of signals conveyed by the VSCT and SRCP to the cerebellum is the information regarding activity of the generator of rhythmic oscillations that is located in the L3-L5 spinal segments.

Polarization of cytoplasmic fragments microsurgically detached from mouse fibroblasts

We have studied the polarity of cytoplasm organization in tiny fragments of mouse embryo fibroblasts, produced by the microsurgical separation of long processes of cytochalasin-treated cells. In the cytochalasin-free medium fragments respread and developed small lamellas at one or both of their ends. Granules, visible at phase-contrast optics, were always collected in the central part of the fragment. Lamellas of the fragment, as well as lamellar cytoplasm of parent cells, were able to clear surface receptors patched by concanavalin A and an antibody to concanavalin A. Immunofluorescence microscopy showed that the fragments always contained actin microfilament bundles parallel to the long axis of the fragment, but microtubules were present not more than for 6 hrs after detachment of the fragments from the cell bodies. Fragments detached from the cells treated with colcemid and cytochalasin simultaneously and transferred into the drug-free medium never had any microtubules. In spite of that, their behaviour was similar to the behaviour of the fragments that were produced from the control cells treated only with cytochalasin. These results show that the small fragments of mouse embryo fibroblasts are able to maintain the polar organization of cytoplasm and the microtubules are not responsible for this organization.