Heparin modulates the organization of hydrated collagen gels and inhibits gel contraction by fibroblasts

We studied the effects of extracellular matrix components on fibroblast contraction of hydrated collagen gels. After 4-h incubations, heparin-containing collagen gels contracted only 10% compared with 50% contraction of control gels. Contraction was not affected by hyaluronic acid, dermatan sulfate, or fibronectin, implying that the activity of heparin was specific. The possibility that heparin inhibited attachment of the cells to the gels was ruled out. Also, addition of heparin to the incubation medium had no effect on contraction. Microscopic examination showed that control collagen gels were composed of a uniform network of interlocking fibrils of similar sizes. Heparin-containing gels, on the other hand, were highly variable with some collagen bundles containing 5-6 collagen fibrils and other regions containing amorphous material. Unlike the control gels, the fibrils of heparin-containing gels were not continuously interconnected. Based on the results, we propose that fibroblasts attach normally to the collagen fibrils of heparin-containing gels and attempt to contract the gels, but the mechanical forces exerted by fibroblasts on individual collagen fibrils cannot be propagated throughout the gels.     

Complementary roles of microtubules and microfilaments in the lung fibroblast-mediated contraction of collagen gels: Dynamics and the influence of cell density

Summary Fibroblasts are important cellular components in wound healing, scar formation, and fibrotic disorders; and the fibroblast-populated collagen-gel (FPCG) model allows examination of fibroblast behavior in an in vitro three-dimensional environment similar to that in vivo. Contraction of free-floating FPCGs depends, on an active and dynamic cytoskeleton, and the contraction dynamics are highly influenced by cell density. We investigated mechanistic differences between high- and low-cell density FPCG contraction by evaluating contraction dynamics in detail, using specific cytoskeletal disruptors. Collagen gels were seeded with human lung fibroblasts at either high (HD) or low (LD) density, and incubated with or without cytoskeletal disruptors colchicine (microtubules) or cytochalasin D (microfilaments). Gel area was measured daily. FPCG contraction curves were essentially sigmoidal, featuring an initial period of no contraction (lag phase), followed by a period of rapid contraction (log phase). Contraction curves of HD-FPCGs were distinct from those of LD-FPCGs. For example, HD-FPCGs had a negligible lag phase (compared with 3 d for LD-FPCGs) and exhibited a higher rate of log-phase contraction. Both colchicine and cytochalasin dose-dependently inhibited contraction but specifically affected different phases of contraction in HD- and LD-FPCGs; and colchicine inhibited LD-FPCGs much more than HD-FPCGs. The data indicate that LD- and HD-FPCGs contract through different primary mechanisms. Microtubules and microfilaments are both complementarily and dynamically involved in the contraction of FPCGs, and cell density influences primary cytoskeletal mechanisms. These results provide valuable information about fibroblast behavior in healing and fibrosis, and may suggest novel treatment options.     

Interaction of microtubules and microfilaments in the zone of distal elongation of Arabidopsis thaliana roots

Investigations on GFP-MAP4 and GFP-ABD2 plants of Arabidopsis thaliana with the usage of inhibitors of actin and tubulin polymerization revealed that stability of cell growth in the zone of distal elongation of a root is provided by structural interactions between microtubules and actin filaments. The experiments with clinorotation and inhibition analysis enabled us to demonstrate that gravitation is a determining factor for the interaction between cytoskeleton elements in the zone of distal elongation in a root.     

The arrangement of microfilaments and microtubules in the periphery of spreading fibroblasts and glial cells

Studies of spreading fibroblasts and glial cells showed that the initial phase of the spreading process on a solid substratum proceeds by sequential development of different kinds of protrusions. Initially there is a high blebbing activity which is followed by development of small lamellipodia and somewhat later microspikes are formed. In the periphery of the spreading cells several types of microfilament organizations are displayed, these seem to be related to different stages in the cycles of extensions and retractions performed by the lamellipodia. The presence of microtubules and their relation to the different microfilament organizations are also shown.     

The role of microtubules and microfilaments in the micronucleus ofParamecium bursaria during mitosis

Summary A unique spindle apparatus develops during mitosis in the micronucleus ofParamecium bursaria. During interphase the micronucleus contains short microtubule profiles and clumps of condensed chromatin. Throughout mitosis the nuclear envelope remains intact. During prophase, cup-shaped structures termed microlamellae develop in close association with regions of condensed chromatin. Each micromella consists of an outer sublamella, an inner sublamellae, and ring-shaped structures termed microsepta that join the two sublamellae. Microtubules elongate parallel to the division axis. During metaphase, the microlamellae appear to act as kinetochorelike structures that aid in the alignment of the chromosomes. The microlamellae appear conical and join to a meshwork of microfilaments at their apices. Further toward the polar regions the microfilaments join with microtubules that converge and terminate near the nuclear envelope. During metaphase-anaphase and anaphase the chromosomes are apparently moved by the microfilaments pulling on the kinetochorelike microlamellae. Also during metaphase-anaphase, extranuclear microtubules join the nuclear envelope of the micronucleus to microtubule elements of the cell cortex. By anaphasetelophase, microlamellae and the microfilament meshwork degenerate and microtubules represent the only spindle elements. The evidence of this report supports the hypothesis that microfilaments can participate with microtubules in the movement of chromosomes.This report is part of a Ph.D. Thesis presented by the senior author at Fordham University.     

The role of microtubules and microfilaments in the hydrosmotic response to antidiuretic hormone

To test the effects of colchicine and cytochalasin B on the ADH-induced response, unidirectional and net water fluxes were measured at one or two minutes intervals in frog urinary bladder. The action of these agents on the appearance of intramembrane particles aggregates in the luminal membrane of target cells under oxytocin stimulation and the changes in the tissue ultrastructure induced by cytochalasin B were also studied. It was observed that: the time-course of the response to oxytocin was strongly slowed by colchicine while the washout was not affected; the time-course of the 'on and off' of the response to oxytocin was not modified by cytochalasin B; cytochalasin B pretreatment proportionally reduced unidirectional and net water fluxes measured after glutaraldehyde fixation; the combined action of colchicine and cytochalasin B proportionally reduced the net water flux and the number of intramembrane particles aggregates, observed in freeze-fracture studies; after cytochalasin B action the dilation of intercellular spaces classically observed under oxytocin stimulation is strongly reduced. It is concluded that: microtubules probably play an important role in the water channels plug-in, but not in their removal; microfilaments integrity is necessary for the mechanisms inducing intercellular space dilation and the observed results confirm that water permeability is controlled by the number of permeation units present in the luminal border of granular cells and probably represented by the intramembrane particle aggregates.     

Behaviour of Duchenne dystrophy fibroblasts in collagen gels

In order to clarify the possible involvement of the cell surface in the pathogenesis of Duchenne muscular dystrophy, we have examined the behaviour of fibroblasts cultured from Duchenne patients in hydrated collagen lattices. No differences could be found between Duchenne and normal skin fibroblasts, either after initial seeding or following prolonged culture within the collagen gel.     

Roles of microfilaments and microtubules in paxillin dynamics

We investigated the roles of microfilaments and microtubules in the localization and tyrosine phosphorylation of paxillin, a focal adhesion-associated signaling molecule, in bovine aortic endothelial cells (BAECs). Paxillin tyrosine phosphorylation is inhibited by cytochalasin D (CD), but slightly increased by colchicine and paclitaxol (taxol). CD also caused an overall disassembly of paxillin-containing focal adhesions (paxillin-FAs) and translocation of paxillin to the cytoplasm and perinuclear region with a diffuse distribution. Meanwhile, colchicine and taxol caused a disassembly of paxillin-FAs from cell periphery and lamellipodia, and their assembly in cell center. These results indicate that actin filaments are important in paxillin assembly in the FAs of the whole ECs and that microtubules are critical in paxillin assembly in cell periphery and lamellipodia; thus the microfilaments and microtubules play differential roles in the dynamics of paxillin assembly/disassembly. Our findings also suggest that tyrosine phosphorylation is an important element in paxillin dynamics at FAs.     

The role of microfilaments and of microtubules in taurocholate uptake by isolated rat liver cells

The role of microfilaments and microtubules on bile salt transport was studied by investigating the influence of a microfilament and a microtubule inhibitor, cytochalasin B and colchicine, respectively, on taurocholate uptake by isolated hepatocytes in vitro. Hepatocytes were prepared by the enzyme perfusion method and [¹⁴C]taurocholate uptake velocity was determined by a filtration assay. Taurocholate uptake obeyed Michaelis-Menten kinetics, maximal uptake velocity and apparent half-saturation constants averaging $\text{0.87 ±}\text{SD}\text{0.05}\text{nmol}\text{· s}{}^{\mathrm{?1}}\text{· 10}{}^{\mathrm{?6}}\text{cells}$ and $\text{10.9 ± 1.8 μ}\text{M}$, respectively. Cytochalasin B ($\text{4.2–420 μ}\text{M}$) inhibited taurocholate uptake in a competitive fashion; $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ being $\text{33 ± 7 μ}\text{M}$. At concentrations above 100 μM the compound decreased ³⁶Cl membrane potential and intracellular K⁺ concentration. Other parameters of cell viability were not affected by cytochalasin B. Colchicine (0.1–1.0 mM), by contrast, inhibited taurocholate uptake non-competitively, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ being $\text{0.47 ± 0.07}\text{mM}$. The inhibition brought about by colchicine was considerably smaller than that induced by cytochalasin B. None of the parameters of cell viability tested was affected by colchicine. These results suggest that microfilaments may be involved in the carrier-mediated hepatocellular transport of bile salts. This could, at least in part, account for cytochalasin B-induced cholestasis. The contribution of the microtubular system, if any, is less important quantitatively. The mechanisms whereby these two components of the cytoskeleton partake in bile salt transport remain to be elucidated.     

Functional role for stable microtubules in lens fiber cell elongation

The process of tissue morphogenesis, especially for tissues reliant on the establishment of a specific cytoarchitecture for their functionality, depends a balanced interplay between cytoskeletal elements and their interactions with cell adhesion molecules. The microtubule cytoskeleton, which has many roles in the cell, is a determinant of directional cell migration, a process that underlies many aspects of development. We investigated the role of microtubules in development of the lens, a tissue where cell elongation underlies morphogenesis. Our studies with the microtubule depolymerizing agent nocodazole revealed an essential function for the acetylated population of stable microtubules in the elongation of lens fiber cells, which was linked to their regulation of the activation state of myosin. Suppressing myosin activation with the inhibitor blebbistatin could attenuate the loss of acetylated microtubules by nocodazole and rescue the effect of this microtubule depolymerization agent on both fiber cell elongation and lens integrity. Our results also suggest that acetylated microtubules impact lens morphogenesis through their interaction with N-cadherin junctions, with which they specifically associate in the region where lens fiber cell elongate. Disruption of the stable microtubule network increased N-cadherin junctional organization along lateral borders of differentiating lens fiber cells, which was prevented by suppression of myosin activity. These results reveal a role for the stable microtubule population in lens fiber cell elongation, acting in tandem with N-cadherin cell-cell junctions and the actomyosin network, giving insight into the cooperative role these systems play in tissue morphogenesis.     

The reorganization of microtubules and microfilaments in differentiating keratinocytes

Using immunofluorescence techniques, we have examined the microtubules and microfilaments in colonies of terminally differentiating human keratinocytes in tissue culture. The undifferentiated keratinocytes contained numerous microtubules, which radiated from a centrosomal organization center (MTOC). Differentiating keratinocytes, which leave the basal layer and begin to synthesize involucrin, displayed an altered cytoskeleton. Thick mats and coils of microtubules formed throughout the cytoplasm of the differentiated squames, and microfilaments were no longer visible after staining with phalloidin. Instead, only scattered stipples of phalloidin-stained material were observed. The results suggest that the terminal differentiation of epidermal cells involves a reorganization not only of the keratin filaments but of the entire cytoskeleton.     

Endothelial cells secrete a factor that promotes fibroblast contraction of hydrated collagen gels

Bovine aortic endothelial cells (BAEC), grown in vitro, are shown to synthesize and secrete factor(s) that stimulate fibroblasts to contract collagen matrices. The amount of contraction-promoting activity in the conditioned media is dependent on conditioning time and the number of cells in the culture. Production of the contraction-promoting activity continues at a high stable level for at least 5 d in serum-free medium but is abolished when the cells are exposed to an inhibitor of protein synthesis. The mechanism of action of the contraction factor(s) derived from endothelial cells was compared with that of unidentified serum factors. The endothelial cell-secreted factor(s) depends on active protein synthesis by the target cell but does not need to be present during the contraction process. The serum factors on the other hand promote collagen contraction in the absence of de novo protein synthesis but need to be continuously present. Preliminary biochemical characterization of the contraction-promoting factors produced by endothelial cells revealed properties similar to those of previously identified growth factors. However, the BAEC-secreted factor was found to be distinct from a previously identified contraction-promoting transforming growth factor beta.     

A possible complementary role of actin microfilaments and microtubules in triacylglycerol secretion by isolated rate hepatocytes

Incubation of isolated rat hepatocytes with phalloidin, cytochalasins (which, respectively, stabilize and destabilize actin microfilaments), or colchicine (which inhibits polymerization of microtubules), resulted in a dose-dependent inhibition of triacyglycerol secretion (an index of very low density lipoprotein secretion). Upon removal of drugs from incubation media, the inhibitory effect of cytochalasin D on triacylglycerol secretion was reversible, while such was not the case for phalloidin. When used at maximal concentrations, the combined presence of phalloidin + colchicine or cytochalasin D + colchicine had additive inhibitory effects upon hepatic triacylglycerol secretion, which was virtually blocked; this was not the case for phalloidin + cytochalasin D. These experiments support the concept that microfilaments and microtubules may have complementary functions for the hepatic secretion of very low density lipoproteins.     

A primary role for microfilaments, but not microtubules, in hormone-induced cytoplasmic retraction

The distributions of microfilaments and microtubules were studied during transient hormone-induced changes in cell shape (retraction-respreading). Two cell types (fibroblasts and bone cells), differentially responsive to parathyroid hormone (PTH) and prostaglandin E₂ (PGE₂), were analysed. The cytoplasm of fibroblasts retracted in response to PGE₂ but not PTH, whereas bone cells could respond to both PGE₂ and PTH. Time-lapse photomicrography indicated that the retraction began within minutes of hormone addition, while respreading occurred over longer times, up to 8 h. Affinity-purified actin and tubulin antibodies were used to follow the appearance of microtubules and microfilaments during both the retraction and the respreading phases. Microtubules appeared not to reorganize noticeably, although they were squeezed closer together in cellular pseudopods; no extensive loss or growth was detectable. Microfilaments did alter drastically their appearance and distributions. Soon after hormone addition when earliest detectable cytoplasmic retraction was evident, microfilament bundles appeared to break down. Remaining microfilament bundles consisted of relatively short, non-aligned fragments or aggregates. During respreading, microfilament bundles regrew and realigned throughout the cytoplasm. These data suggest a primary role for microfilaments, but probably not microtubules, in these cell shape changes.