The role of actin filaments and microtubules in hepatocyte spheroid self-assembly

Cultured rat hepatocytes self-assemble into three-dimensional structures or spheroids that exhibit ultrastructural characteristics of native hepatic tissue and enhanced liver-specific functions. The spheroid formation process involves cell translocation and changes in cell shape, indicative of the reorganization of the cytoskeletal elements. To elucidate the function of the cytoskeleton, hepatocytes undergoing spheroid formation were treated with drugs that disrupt the different cytoskeletal components. Cytochalasin D, which targets the actin filaments, caused inhibition of spheroid formation. The role of microtubules in this process was assessed by incubating the cells with taxol or nocodazole. Perturbation of microtubules had minimal effects on spheroid assembly. Scanning electron micrographs showed no morphological differences between spheroids formed in control cultures and those formed in the presence of taxol or nocodazole. In addition, the effects of those agents on hepatocyte functions were investigated. Albumin secretion and cytochrome P450 2B1/2 activities of hepatocytes were comparable in spheroids formed in the presence of taxol or nocodazole to those formed in control cultures. The levels of these liver-specific activities were lower in cytochalasin D--treated cultures where only dispersed cells or cell clumps were found but spheroids had not found. Thus, hepatocytes require an intact actin network to self-assemble efficiently into functional tissue-like structures. Perturbation of the microtubule lattice does not impair the formation process. Events that transpire during hepatocyte spheroid self-assembly exhibit striking similarities to processes commonly observed in tissue morphogenesis. The results provide insight into the mechanisms that cells employ to organize into tissues and can contribute to our understanding of how to control the cellular assembly in tissue engineering and clinical applications.     

Centrosome separation: respective role of microtubules and actin filaments

In mammalian cells, the separation of centrosomes is a prerequisite for bipolar mitotic spindle assembly. We have investigated the respective contribution of the two cytoskeleton components, microtubules and actin filaments, in this process. Distances between centrosomes have been measured during cell cycle progression in Xenopus laevis XL2 cultured cells in the presence or absence of either network. We considered two stages in centrosome separation: the splitting stage, when centrosomes start to move apart (minimum distance of 1 microm), and the elongation stage (from 1 to 7 microm). In interphase, depolymerisation of microtubules by nocodazole significantly inhibited the splitting stage, while the elongation stage was, on the contrary, facilitated. In mitosis, while nocodazole treatment completely blocked spindle assembly, in prophase, we observed that 55% of the centrosomes separated, versus 94% in the control. Upon actin depolymerisation by latrunculin, splitting of the interphase centrosome was blocked, and cells entered mitosis with unseparated centrosomes. Cells compensated for this separation delay by increasing the length of both prophase and prometaphase stages to allow for centrosome separation until a minimal distance was reached. Then the cells passed through anaphase, performing proper chromosome separation, but cytokinesis did not occur, and binuclear cells were formed. Our results clearly show that the actin microfilaments participate in centrosome separation at the G2/M transition and work in synergy with the microtubules to accelerate centrosome separation during mitosis.     

The role of palladin in actin organization and cell motility

Palladin is a widely expressed protein found in stress fibers, focal adhesions, growth cones, Z-discs, and other actin-based subcellular structures. It belongs to a small gene family that includes the Z-disc proteins myopalladin and myotilin, all of which share similar Ig-like domains. Recent advances have shown that palladin shares with myotilin the ability to bind directly to F-actin, and to crosslink actin filaments into bundles, in vitro. Studies in a variety of cultured cells suggest that the actin-organizing activity of palladin plays a central role in promoting cell motility. Correlative evidence also supports this hypothesis, as palladin levels are typically up-regulated in cells that are actively migrating: in developing vertebrate embryos, in cells along a wound edge, and in metastatic cancer cells. Recently, a mutation in the human palladin gene was implicated in an unusually penetrant form of inherited pancreatic cancer, which has stimulated new ideas about the role of palladin in invasive cancer.     

Identification of motifs of Burkholderia pseudomallei BimA required for intracellular motility, actin binding, and actin polymerization

Actin-based motility of the melioidosis pathogen Burkholderia pseudomallei requires BimA (Burkholderia intracellular motility A). The mechanism by which BimA mediates actin assembly at the bacterial pole is ill-defined. Toward an understanding of the regions of B. pseudomallei BimA required for intracellular motility and the binding and polymerization of actin, we constructed plasmid-borne bimA variants and glutathione-S-transferase fusion proteins with in-frame deletions of specific motifs. A 13-amino-acid direct repeat and IP₇ proline-rich motif were dispensable for actin binding and assembly in vitro, and expression of the mutated proteins in a B. pseudomallei bimA mutant restored actin-based motility in J774.2 murine macrophage-like cells. However, two WASP homology 2 (WH2) domains were found to be required for actin binding, actin assembly, and plaque formation. A tract of five PDASX direct repeats influenced the polymerization of pyrene-actin monomers in vitro and was required for actin-based motility and intercellular spread, but not actin binding. None of the mutations impaired surface expression or polar targeting of BimA. The number of PDASX repeats varied in natural isolates from two to seven. Such repeats acted additively to promote pyrene-actin polymerization in vitro, with stepwise increases in the rate of polymerization as the number of repeats was increased. No differences in the efficiency of actin tail formation could be discerned between strains expressing BimA variants with two, five, or seven PDASX repeats. The data provide valuable new insights into the role of conserved and variable motifs of BimA in actin-based motility and intercellular spread of B. pseudomallei.     

A crucial role for profilin-actin in the intracellular motility of Listeria monocytogenes

We have examined the effect of covalently crosslinked profilin–actin (PxA), which closely matches the biochemical properties of ordinary profilin–actin and interferes with actin polymerization in vitro and in vivo, on Listeria monocytogenes motility. PxA caused a marked reduction in bacterial motility, which was accompanied by the detachment of bacterial tails. The effect of PxA was dependent on its binding to proline-rich sequences, as shown by the inability of P_H133SxA, which cannot interact with such sequences, to impair Listeria motility. PxA did not alter the motility of a Listeria mutant that is unable to recruit Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) proteins and profilin to its surface. Finally, PxA did not block the initiation of actin-tail formation, indicating that profilin–actin is only required for the elongation of actin filaments at the bacterial surface. Our findings provide further evidence that profilin–actin is important for actin-based processes, and show that it has a key function in Listeria motility.     

The role of actin and microtubule networks in plasmid DNA intracellular trafficking

This work is focused on the function of the microtubule and actin networks in plasmid DNA transport during liposomal transfection. We observed strong binding of plasmid DNA-lipid complexes (lipoplexes) to both networks and directional long-range motion of these lipoplexes along the microtubules. Disruption of either of these networks led to the cessation of plasmid transport to the nucleus, a decreased mobility of plasmids, and accumulation of plasmid DNA in large aggregates at the cell periphery. Our findings show an indispensable but different role of both types of cytoskeleton, actin and microtubular, in the processes of gene delivery.     

Receptor-mediated actin assembly in electropermeabilized neutrophils: role of intracellular pH

Neutrophil activation by a variety of stimuli is accompanied by an intracellular acidification, which has been postulated to mediate actin polymerization (Yuli and Oplatka, Science 1987, 235, 340). This hypothesis was tested using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin staining and flow cytometry, or right angle light scattering to study actin assembly in intact and electrically permeabilized human neutrophils. Intracellular pH was measured fluorimetrically using a pH sensitive dye. In cells stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) at 21 degrees C, actin assembly clearly preceded the intracellular acidification in response to fMLP. Moreover, actin polymerization persisted in cells where intracellular pH was clamped near the resting (unstimulated) level using nigericin/K+. Finally, fMLP induced a significant increase in F-actin content in electropermeabilized neutrophils equilibrated with an extracellular medium containing up to 50 mM HEPES. These observations indicate that fMLP-stimulated F-actin assembly is not mediated by a decrease in intracellular pH and suggest that changes in transmembrane potential and ionic gradients are unlikely to mediate actin polymerization.     

Pivotal role of actin depolymerization in the regulation of cochlear outer hair cell motility

Cochlear outer hair cells undergo reversible changes in shape when externally stimulated. This response, known as OHC motility, is a central component of the cochlear amplifier, the mechanism responsible for the high sensitivity of mammalian hearing. We report that actin depolymerization, as regulated by activation/inhibition of LIMK/cofilin-mediated pathways, has a pivotal role in OHC motility. LIMK-mediated cofilin phosphorylation, which inhibits the actin depolymerizing activity of this protein, increases both electromotile amplitude and total length of guinea pig OHCs. In contrast, a decrease in cofilin phosphorylation reduces both OHC electromotile amplitude and OHC length. Experiments with acetylcholine and lysophosphatidic acid indicate that the effects of these agents on OHC motility are associated with regulation of cofilin phosphorylation via different signaling cascades. On the other hand, nonlinear capacitance measurements confirmed that all observed changes in OHC motile response were independent of the performance of the motor protein prestin. Altogether, these results strongly support the hypothesis that the cytoskeleton has a major role in the regulation of OHC motility, and identify actin depolymerization as a key process for modulating cochlear amplification.     

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.     

The arresting action of microtubules in cell motility

Comment on: Ganguly A, et al. Oncotarget 2011; 2:368-77.     

Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility

The Listeria monocytogenes ActA protein mediates actin-based motility by recruiting and stimulating the Arp2/3 complex. In vitro, the actin monomer-binding region of ActA is critical for stimulating Arp2/3-dependent actin nucleation; however, this region is dispensable for actin-based motility in cells. Here, we provide genetic and biochemical evidence that vasodilator-stimulated phosphoprotein (VASP) recruitment by ActA can bypass defects in actin monomer-binding. Furthermore, purified VASP enhances the actin-nucleating activity of wild-type ActA and the Arp2/3 complex while also reducing the frequency of actin branch formation. These data suggest that ActA stimulates the Arp2/3 complex by both VASP-dependent and -independent mechanisms that generate distinct populations of actin filaments in the comet tails of L. monocytogenes. The ability of VASP to contribute to actin filament nucleation and to regulate actin filament architecture highlights the central role of VASP in actin-based motility.     

Possible role of non-classical chromatophorotropins on the regulation of the crustacean erythrophore.

Two neuropeptides, the pigment dispersing hormone (PDH) and the pigment concentrating hormone (PCH), are well known to respectively promote centrifugal and centripetal granule translocation in the freshwater shrimp Macrobrachium potiuna erythrophores. Herein, we demonstrate for the first time the effects of crustacean non-classical chromatophorotropins on the pigment migration in M. potiuna erythrophores. Although proctolin, 20-hydroxyecdisone (20HE), and melatonin were ineffective, the crustacean cardioactive peptide (CCAP) was a full agonist, inducing pigment dispersion in a dose-dependent manner with EC(50) of 9.5. 10(-7) M. In addition, concentrations of CCAP lower than the minimal effective dose (10(-8) and 10(-7) M) decreased the PCH-induced aggregation, shifting rightward the dose-response curve (DRC) to PCH 2.2- and 29-fold, respectively. Surprisingly, melatonin (10(-7) and 10(-6) M) also shifted to the right 8.7- and 46.5-fold, respectively, the DRC to PCH. In conclusion, our data demonstrate that besides PCH and PDH, CCAP and melatonin also regulate the pigment migration within the crustacean erythrophore. J. Exp. Zool. 284:711-716, 1999.Copyright 1999 Wiley-Liss, Inc.     

A critical role of tropomyosins in TGF-beta regulation of the actin cytoskeleton and cell motility in epithelial cells

We have investigated transforming growth factor beta (TGF-beta)-mediated induction of actin stress fibers in normal and metastatic epithelial cells. We found that stress fiber formation requires de novo protein synthesis, p38Mapk and Smad signaling. We show that TGF-beta via Smad and p38Mapk up-regulates expression of actin-binding proteins including high-molecular-weight tropomyosins, alpha-actinin and calponin h2. We demonstrate that, among these proteins, tropomyosins are both necessary and sufficient for TGF-beta induction of stress fibers. Silencing of tropomyosins with short interfering RNAs (siRNAs) blocks stress fiber assembly, whereas ectopic expression of tropomyosins results in stress fibers. Ectopic-expression and siRNA experiments show that Smads mediate induction of tropomyosins and stress fibers. Interestingly, TGF-beta induction of stress fibers was not accompanied by changes in the levels of cofilin phosphorylation. TGF-beta induction of tropomyosins and stress fibers are significantly inhibited by Ras-ERK signaling in metastatic breast cancer cells. Inhibition of the Ras-ERK pathway restores TGF-beta induction of tropomyosins and stress fibers and thereby reduces cell motility. These results suggest that induction of tropomyosins and stress fibers play an essential role in TGF-beta control of cell motility, and the loss of this TGF-beta response is a critical step in the acquisition of metastatic phenotype by tumor cells.     

RhoA regulates peroxisome association to microtubules and the actin cytoskeleton

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.