Cytoskeleton and morphogenesis in brown algae

BACKGROUND: Morphogenesis on a cellular level includes processes in which cytoskeleton and cell wall expansion are strongly involved. In brown algal zygotes, microtubules (MTs) and actin filaments (AFs) participate in polarity axis fixation, cell division and tip growth. Brown algal vegetative cells lack a cortical MT cytoskeleton, and are characterized by centriole-bearing centrosomes, which function as microtubule organizing centres. SCOPE: Extensive electron microscope and immunofluorescence studies of MT organization in different types of brown algal cells have shown that MTs constitute a major cytoskeletal component, indispensable for cell morphogenesis. Apart from participating in mitosis and cytokinesis, they are also involved in the expression and maintenance of polarity of particular cell types. Disruption of MTs after Nocodazole treatment inhibits cell growth, causing bulging and/or bending of apical cells, thickening of the tip cell wall, and affecting the nuclear positioning. Staining of F-actin using Rhodamine-Phalloidin, revealed a rich network consisting of perinuclear, endoplasmic and cortical AFs. AFs participate in mitosis by the organization of an F-actin spindle and in cytokinesis by an F-actin disc. They are also involved in the maintenance of polarity of apical cells, as well as in lateral branch initiation. The cortical system of AFs was found related to the orientation of cellulose microfibrils (MFs), and therefore to cell wall morphogenesis. This is expressed by the coincidence in the orientation between cortical AFs and the depositing MFs. Treatment with cytochalasin B inhibits mitosis and cytokinesis, as well as tip growth of apical cells, and causes abnormal deposition of MFs. CONCLUSIONS: Both the cytoskeletal elements studied so far, i.e. MTs and AFs are implicated in brown algal cell morphogenesis, expressed in their relationship with cell wall morphogenesis, polarization, spindle organization and cytokinetic mechanism. The novelty is the role of AFs and their possible co-operation with MTs.     

Switching of membrane organelles between cytoskeletal transport systems is determined by regulation of the microtubule-based transport

Intracellular transport of membrane organelles occurs along microtubules (MTs) and actin filaments (AFs). Although transport along each type of the cytoskeletal tracks is well characterized, the switching between the two types of transport is poorly understood because it cannot be observed directly in living cells. To gain insight into the regulation of the switching of membrane organelles between the two major transport systems, we developed a novel approach that combines live cell imaging with computational modeling. Using this approach, we measured the parameters that determine how fast membrane organelles switch back and forth between MTs and AFs (the switching rate constants) and compared these parameters during different signaling states. We show that regulation involves a major change in a single parameter: the transferring rate from AFs onto MTs. This result suggests that MT transport is the defining factor whose regulation determines the choice of the cytoskeletal tracks during the transport of membrane organelles.     

Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets

The focal adhesion protein VASP, a possible link between signal transduction pathways and the microfilament system, is phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro and in intact cells. Here, the analysis of VASP dephosphorylation by the serine/threonine protein phosphatases (PP) PP1, PP2A, PP2B and PP2C in vitro is reported. The phosphatases differed in their selectivity with respect to the dephosphorylation of individual VASP phosphorylation sites. Incubation of human platelets with okadaic acid, a potent inhibitor of PP1 and PP2A, caused the accumulation of phosphorylated VASP indicating that the phosphorylation status of VASP in intact cells is regulated to a major extent by serine/ threonine protein phosphatases. Furthermore, the accumulation of phosphorylated cAMP-dependent protein kinase substrate(s) appears to account for inhibitory effects of okadaic acid on platelet function.     

Involvement of protein phsophatase-1 in cytoskeletal organization of cultured endothelial cells

The phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effect of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vien endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non-selective kinase inhibitor, K-252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP-1. The catalytic subunit of PP-1 was also identified in HUVECs by Western blotting with its monoclonal antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton. © 1995 Wiley-Liss, Inc.     

Isolation of a novel 190 kDa protein from tobacco BY-2 cells: possible involvement in the interaction between actin filaments and microtubules

Interaction between actin filaments (AFs) and microtubules (MTs) has been reported in various plant cells, and the presence of a factor(s) connecting these two cytoskeletal networks has been suggested, but its molecular entity has not been elucidated yet. We obtained a fraction containing MT-binding polypeptides, which induced bundling of AFs and of MTs. A 190 kDa polypeptide which associated with AFs was selectively isolated from the fraction. This polypeptide was thought to have an ability to bind to both AFs and MTs. We raised a monoclonal antibody against the 190 kDa polypeptide. Immunostaining demonstrated the association of the 190 kDa polypeptide with AF bundles and with MT bundles formed in vitro. Immunocytochemical studies throughout the cell cycle revealed that the 190 kDa polypeptide was localized in the nucleus before nuclear envelope breakdown, and in the spindle and the phragmoplast during cell division. After the re-formation of the nuclear envelope, the 190 kDa polypeptide was sequestered to the daughter nuclei. Using the antibody, we succeeded in cloning a cDNA encoding the 190 kDa polypeptide.     

Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis

In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells.     

Sites of actin filament initiation and reorganization in cold-treated tobacco cells

Cytoskeletal proteins assemble into dynamic polymers that play many roles in nuclear and cell division, signal transduction, and determination of cell shape and polarity. The distribution and dynamics of microtubules (MTs) and actin filaments (AFs) are determined, among other factors, by the location of their nucleation sites. Whereas the sites of microtubule nucleation in plants are known to be located under the plasma membrane and on the nuclear envelope during interphase, there is a striking lack of information about nucleation sites of AFs. In the studies reported herein, low temperature (0 °C) was used to de‐polymerize AFs and MTs in tobacco BY‐2 (Nicotiana tabacum L.) cells at interphase. The extent of de‐polymerization of cytoskeletal filaments in interphase cells during cold treatment and the subcellular distribution of nucleation sites during subsequent recovery at 25 °C were monitored by means of fluorescence microscopy. The results show that AFs re‐polymerized rapidly from sites located in the cortical region and on the nuclear envelope, similarly to the initiation sites of MTs. In contrast to MTs, however, complete reconstitution of AFs was preceded by the formation of transient actin structures including actin dots, rods, and filaments with a dotted signal. Immunoblotting of soluble and sedimentable protein fractions showed no changes in the relative amounts of free and membrane‐bound actin or tubulin.     

Actin-associated Proteins in the Pathogenesis of Podocyte Injury

Podocytes have a complex cellular architecture with interdigitating processes maintained by a precise organization of actin filaments. The actin-based foot processes of podocytes and the interposed slit diaphragm form the final barrier to proteinuria. The function of podocytes is largely based on the maintenance of the normal foot process structure with actin cytoskeleton. Cytoskeletal dynamics play important roles during normal podocyte development, in maintenance of the healthy glomerular filtration barrier, and in the pathogenesis of glomerular diseases. In this review, we focused on recent findings on the mechanisms of organization and reorganization of these actin-related molecules in the pathogenesis of podocyte injury and potential therapeutics targeting the regulation of actin cytoskeleton in podocytopathies.     

Identification, purification, and characterization of a novel serine/threonine protein phosphatase from bovine brain.

A novel serine/threonine protein phosphatase is identified, and the catalytic subunit, obtained from a detergent extraction of the pellet generated by a 100,000 x g centrifugation of a whole bovine brain homogenate, is purified and characterized. The protein phosphatase, designated as PP3, has a Mr of 36,000, does not require divalent cations for activity, is stimulated rather than inhibited by inhibitor 2, is inhibited by both okadaic acid and microcystin-LR with an intermediate IC50 compared to type 1 and type 2A protein phosphatases, and preferentially dephosphorylates the beta subunit of phosphorylase kinase. Substrate specificity, immunoblotting with type-specific antisera, and the amino acid sequences of peptides derived from PP3 indicate that PP3 is not an isoform of any known serine/threonine protein phosphatase.     

Protein phosphatase 2A regulates apoptosis in intestinal epithelial cells

Polyamine depletion prevents apoptosis by increasing serine/threonine phosphorylation leading to either inactivation or activation of pro- and anti-apoptotic proteins, respectively. Despite evidence that protein kinases are regulators of apoptosis, a specific role for protein phosphatases in regulating cell survival has not been established. In this study, we show that polyamine depletion inhibits serine/threonine phosphatase 2A (PP2A). Inhibition of PP2A in cells depleted of polyamines correlated well with increased phosphorylation of Bad at Ser112. Bad Ser112 phosphorylation in response to tumor necrosis factor (TNF)-alpha treatment decreased with time in cells grown in control as well as those grown in the presence of alpha-difluoromethylornithine plus putrescine. However, a sustained increase in the levels of Bad Ser112 phosphorylation was maintained in response to TNF-alpha treatment in cells grown in the presence of alpha-difluoromethylornithine. Inhibition of PP2A by okadaic acid and fostriecin or PP2A small interfering RNA transfection significantly decreased TNF-alpha-induced apoptosis in control and polyamine-depleted cells. Inhibition of PP2A by okadaic acid: 1) increased Bad and Bcl-2 phosphorylation at Ser112 and Ser70, respectively; 2) increased ERK activity; 3) prevented JNK activation; 4) prevented cytochrome c release, and activation of caspases-9 and -3 in response to TNF-alpha. Inhibition of MEK1 by U0126 prevented phosphorylation of Bad at Ser112. These results indicate that polyamines regulate PP2A activity, and inhibition of PP2A in response to polyamine depletion increases steady state levels of Bad and Bcl-2 proteins and their phosphorylation and thereby prevents cytochrome c release, caspase-9, and caspase-3 activation.     

Microcystin-LR and okadaic acid-induced cellular effects: a dualistic response

Microcystins, potent heptapeptide hepatotoxins produced by certain bloom-forming cyanobacteria, are strong protein phosphatase inhibitors. They covalently bind the serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A), thereby influencing regulation of cellular protein phosphorylation. The paralytic shellfish poison, okadaic acid, is also a potent inhibitor of these PPs. Inhibition of PP1 and PP2A has a dualistic effect on cells exposed to okadaic acid or microcystin-LR, with both apoptosis and increased cellular proliferation being reported. This review summarises the existing data on the molecular effects of microcystin-LR inhibition of PP1 and PP2A both in vivo and in vitro, and where possible, compares this to the action of okadaic acid.     

Nonuniform Microtubular Polarity Established by CHO1/MKLP1 Motor Protein Is Necessary for Process Formation of Podocytes

Podocytes are unique cells that are decisively involved in glomerular filtration. They are equipped with a complex process system consisting of major processes and foot processes whose function is insufficiently understood (Mundel, P., and W. Kriz. 1995. Anat. Embryol. 192:385–397). The major processes of podocytes contain a microtubular cytoskeleton. Taking advantage of a recently established cell culture system for podocytes with preserved ability to form processes (Mundel, P., J. Reiser, A. Zúñiga Mejía Borja, H. Pavenstädt, G.R. Davidson, W. Kriz, and R. Zeller. 1997b. Exp. Cell Res. 36:248–258), we studied the functional significance of the microtubular system in major processes. The following data were obtained: (a) Microtubules (MTs) in podocytes show a nonuniform polarity as revealed by hook-decoration. (b) CHO1/ MKLP1, a kinesin-like motor protein, is associated with MTs in podocytes. (c) Treatment of differentiating podocytes with CHO1/MKLP1 antisense oligonucleotides abolished the formation of processes and the nonuniform polarity of MTs. (d) During the recovery from taxol treatment, taxol-stabilized (nocodazole- resistant) MT fragments were distributed in the cell periphery along newly assembled nocodazole-sensitive MTs. A similar distribution pattern of CHO1/MKLP1 was found under these circumstances, indicating its association with MTs. (e) In the recovery phase after complete depolymerization, MTs reassembled exclusively at centrosomes. Taken together, these findings lead to the conclusion that the nonuniform MT polarity in podocytes established by CHO1/MKLP1 is necessary for process formation.     

Switching between microtubule- and actin-based transport systems in melanophores is controlled by cAMP levels

BACKGROUND: Intracellular transport involves the movement of organelles along microtubules (MTs) or actin filaments (AFs) by means of opposite-polarity MT motors or actin-dependent motors of the myosin family. The correct delivery of organelles to their different destinations involves a precise coordination of the two transport systems. Such coordination could occur through regulation of the densities of the two cytoskeletal systems or through regulation of the activities of the cytoskeletal motors by signaling mechanisms. RESULTS: To investigate the mechanisms of switching between MT and AF-dependent transport, we examine the influence of the densities of the MT and AF network on pigment transport in fish melanophores. We also change signaling by using activators and inhibitors of Protein Kinase A (PKA). We find that the key parameters characterizing pigment granule transport along MTs do not depend on MT density and are not significantly altered by complete disruption of AFs. In contrast, the kinetics of changes in these parameters correlate with the kinetics of changes in the intracellular levels of cAMP and are affected by the inhibitors of PKA, suggesting the regulation of MT- and AF-dependent motors by cAMP-induced signaling. Furthermore, perturbation of cAMP levels prevents the transfer of pigment granules from MTs onto AFs. CONCLUSIONS: We conclude that the switching of pigment granules between the two major cytoskeletal systems is independent of the densities of MT or AF but is tightly controlled by signaling events.     

Inhibitors of protein phosphatases 1 and 2A differentially prevent intrinsic and extrinsic apoptosis pathways

Inhibitors of serine/threonine protein phosphatases can inhibit apoptosis. We investigated which protein phosphatases are critical for this protection using calyculin A, okadaic acid, and tautomycin. All three phosphatase inhibitors prevented anisomycin-induced apoptosis in leukemia cell models. In vitro, calyculin A does not discriminate between PP1 and PP2A, while okadaic acid and tautomycin are more selective for PP2A and PP1, respectively. Increased phosphorylation of endogenous marker proteins was used to define concentrations that inhibited each phosphatase in cells. Concentrations of each inhibitor that prevented anisomycin-induced apoptosis correlated with inhibition of PP2A. The inhibitors prevented Bax translocation to mitochondria, indicating inhibition upstream of mitochondria. Tautomycin and calyculin A, but not okadaic acid, also prevented apoptosis induced through the CD95/Fas death receptor, and this protection correlated with inhibition of PP1. The inhibitors prevented Fas receptor oligomerization, FADD recruitment, and caspase 8 activation. The differential effects of PP1 and PP2A in protection from death receptor and mitochondrial-mediated pathways of death, respectively, may help one to define critical steps in each pathway, and regulatory roles for serine/threonine phosphatases in apoptosis.     

Serine/threonine protein phosphatases PP1 and PP2A are key players in apoptosis

The reversible phosphorylation of proteins controlled by protein kinases and protein phosphatases is a major mechanism that regulates a wide variety of cellular processes. In contrast to C. elegans, recent studies in mammalian cells have highlighted a major role of serine/threonine protein phosphorylation in apoptosis. To illustrate the importance of dephosphorylation processes in apoptosis, this review will focus on recent studies suggesting that the interaction of the serine/threonine protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) with certain regulators of the Bcl-2 family is critically involved in the control of apoptosis.     

Confluence induced threonine41/serine45 phospho-beta-catenin dephosphorylation via ceramide-mediated activation of PP1cgamma

It was previously observed that cell confluence induced up-regulation of neutral sphingomyelinase 2 (nSMase2) and increased ceramide levels [Marchesini N., Osta W., Bielawski J., Luberto C., Obeid L.M. and Hannun Y.A. (2004) J. Biol. Chem., 279, 25101-11]. In this study, we show that, in MCF7 cells, confluence induces the dephosphorylation of phosphorylated-beta-catenin at threonine41/serine45. The effect of confluence on beta-catenin dephosphorylation was prevented by down regulation of nSMase2 using siRNA; reciprocally, exogenous addition of short or very long chain ceramides induced dephosphorylation of beta-catenin. The serine/threonine protein phosphatase inhibitors calyculin A and okadaic acid prevented beta-catenin dephosphorylation during confluence. The specific phosphatase involved was determined by studies using siRNA against the major serine/threonine phosphatases, and the results showed that a specific siRNA against PP1cgamma prevented dephosphorylation of beta-catenin. Moreover, exogenous ceramides and confluence were found to induce the translocation of PP1cgamma to the plasma membrane. All together these results establish: A) a specific intracellular pathway involving the activation of PP1 to mediate the effects of confluence-induced beta-catenin dephosphorylation and B) PP1 as a lipid-regulated protein phosphatase downstream of nSMase2/ceramide. Finally, evidence is provided for a role for this pathway in regulating cell motility during confluence.     

Importance of the C28-C38 hydrophobic domain of okadaic acid for potent inhibition of protein serine-threonine phosphatases 1 and 2A

Okadaic acid is a potent inhibitor of select serine/threonine protein phosphatases. The importance of the C28-C38 hydrophobic domain of okadaic acid for inhibition of PP1 and PP2A was investigated. The hydrophobic domain is required but not sufficient for potent inhibition, and it also contributes to differential inhibition between PP1 and PP2A.     

AlphaV-integrins mediate the mechanoprotective action of osteopontin in podocytes

Increased mechanical load in podocytes due to glomerular hypertension is one of the important factors leading to podocyte damage and chronic kidney disease. In previous studies, we have shown that mechanical stretch increases osteopontin (OPN) expression in podocytes and that exogenous OPN is mechanoprotective via facilitating cytoskeletal reorganization of podocytes. In the present study, we asked whether the mechanoprotective effect of OPN in podocytes is mediated through specific integrins and whether endogenous OPN of podocytes is required for mechanoprotection. Conditionally immortalized mouse podocytes and primary podocytes (PP) from OPN-/- and OPN+/+ mice were used. Cyclic biaxial mechanical stretch (0.5 Hz, 7% linear strain) was applied for up to 3 days. Stretch-induced cell loss was ～30% higher in OPN-/- PP compared with OPN+/+ PP. Increased cell loss of OPN-/- PP was rescued by OPN coating. Analysis of integrin expression by RT-PCR, application of RGD and SLAYGLR peptides and anti-integrin antibodies, small-interfering RNA knockdown of integrins, and application of kinase inhibitors identified αV-integrins (αVβ1, αVβ3, and αVβ5) to mediate the mechano-protective effect of OPN in podocytes involving focal adhesion kinase, Src, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase. Our results demonstrate that endogenous OPN of podocytes plays a nonredundant role in podocyte adaptation to mechanical stretch, and that OPN signaling via α(V)-integrins may represent a relevant therapeutical target in podocytes.     

Confluence induced threonine41/serine45 phospho-β-catenin dephosphorylation via ceramide-mediated activation of PP1cγ

It was previously observed that cell confluence induced up-regulation of neutral sphingomyelinase 2 (nSMase2) and increased ceramide levels [Marchesini N., Osta W., Bielawski J., Luberto C., Obeid L.M. and Hannun Y.A. (2004) J. Biol. Chem., 279, 25101–11]. In this study, we show that, in MCF7 cells, confluence induces the dephosphorylation of phosphorylated-β-catenin at threonine41/serine45. The effect of confluence on β-catenin dephosphorylation was prevented by down regulation of nSMase2 using siRNA; reciprocally, exogenous addition of short or very long chain ceramides induced dephosphorylation of β-catenin. The serine/threonine protein phosphatase inhibitors calyculin A and okadaic acid prevented β-catenin dephosphorylation during confluence. The specific phosphatase involved was determined by studies using siRNA against the major serine/threonine phosphatases, and the results showed that a specific siRNA against PP1cγ prevented dephosphorylation of β-catenin. Moreover, exogenous ceramides and confluence were found to induce the translocation of PP1cγ to the plasma membrane. All together these results establish: A) a specific intracellular pathway involving the activation of PP1 to mediate the effects of confluence-induced β-catenin dephosphorylation and B) PP1 as a lipid-regulated protein phosphatase downstream of nSMase2/ceramide. Finally, evidence is provided for a role for this pathway in regulating cell motility during confluence.     

Contribution of actin filaments and microtubules to quasi-in situ tensile properties and internal force balance of cultured smooth muscle cells on a substrate

The effects of actin filaments (AFs) and microtubules (MTs) on quasi-in situ tensile properties and intracellular force balance were studied in cultured rat aortic smooth muscle cells (SMCs). A SMC cultured on substrates was held using a pair of micropipettes, gradually detached from the substrate while maintaining in situ cell shape and cytoskeletal integrity, and then stretched up to approximately 15% and unloaded three times at the rate of 1 mum every 5 s. Cell stiffness was approximately 20 nN per percent strain in the untreated case and decreased by approximately 65% and approximately 30% following AF and MT disruption, respectively. MT augmentation did not affect cell stiffness significantly. The roles of AFs and MTs in resisting cell stretching and shortening were assessed using the area retraction of the cell upon noninvasive detachment from thermoresponsive gelatin-coated dishes. The retraction was approximately 40% in untreated cells, while in AF-disrupted cells it was <20%. The retraction increased by approximately 50% and decreased by approximately 30% following MT disruption and augmentation, respectively, suggesting that MTs resist intercellular tension generated by AFs. Three-dimensional measurements of cell morphology using confocal microscopy revealed that the cell volume remained unchanged following drug treatment. A concomitant increase in cell height and decrease in cell area was observed following AF disruption and MT augmentation. In contrast, MT disruption significantly reduced the cell height. These results indicate that both AFs and MTs play crucial roles in maintaining whole cell mechanical properties of SMCs, and that while AFs act as an internal tension generator, MTs act as a tension reducer, and these contribute to intracellular force balance three dimensionally.