Macrophage podosomes assemble at the leading lamella by growth and fragmentation

Podosomes are actin- and fimbrin-containing adhesions at the leading edge of macrophages. In cells transfected with beta-actin-ECFP and L-fimbrin-EYFP, quantitative four-dimensional microscopy of podosome assembly shows that new adhesions arise at the cell periphery by one of two mechanisms; de novo podosome assembly, or fission of a precursor podosome into daughter podosomes. The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, fimbrin, integrin, and is in close apposition to the substratum. Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures. Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.     

Cytokine G-protein signaling crosstalk in cardiomyocytes: Attentuation of Jak-STAT activation by endothelin-1

Cytoskeletal reorganization has been shown to participate in cellular remodeling and in the alterations of mechanical function of isolated cardiomyocytes during pressure overload hypertrophy. Post-translational modifications of tubulin towards stabilization of microtubules have also been described in animal models of compensatory hypertrophy, but the status of the microtubules network in end stage heart failure is not clearly established. Using a rat model of congestive heart failure (CHF) induced by aortic banding, we studied the expression of - and -tubulin, as well as their post-translational modification and distribution in the soluble and polymerized fraction by immunoblotting. We found an accumulation of - and -tubulin protein content specifically in the soluble fraction with no change in the polymerized fraction. Amongst the several variants of -tubulin examined, only detyrosinated Glu-tubulin and deglutamylated ₂-tubulin levels were selectively increased during heart failure. Glu-tubulin accumulated in the polymerized fraction while ₂-tubulin levels were increased in the soluble fraction in CHF hearts. These results show that a profound remodeling of the microtubule network occurs in heart failure. This remodeling suggests an increase in the stability of the microtubule network which is discussed in terms of possible functional consequences.     

Inhibition of tubulin assembly and covalent binding to microtubular protein by valproic acid glucuronide in vitro

Acyl glucuronides are reactive metabolites of carboxylate drugs, able to undergo a number of reactions in vitro and in vivo, including isomerization via intramolecular rearrangement and covalent adduct formation with proteins. The intrinsic reactivity of a particular acyl glucuronide depends upon the chemical makeup of the drug moiety. The least reactive acyl glucuronide yet reported is valproic acid acyl glucuronide (VPA-G), which is the major metabolite of the antiepileptic agent valproic acid (VPA). In this study, we showed that both VPA-G and its rearrangement isomers (iso-VPA-G) interacted with bovine brain microtubular protein (MTP, comprised of 85% tubulin and 15% microtubule associated proteins {MAPs}). MTP was incubated with VPA, VPA-G and iso-VPA-G for 2 h at room temperature and pH 7.5 at various concentrations up to 4 mM. VPA-G and iso-VPA-G caused dose-dependent inhibition of assembly of MTP into microtubules, with 50% inhibition (IC₅₀) values of 1.0 and 0.2 mM respectively, suggesting that iso-VPA-G has five times more inhibitory potential than VPA-G. VPA itself did not inhibit microtubule formation except at very high concentrations (≥2 mM). Dialysis to remove unbound VPA-G and iso-VPA-G (prior to the assembly assay) diminished inhibition while not removing it. Comparison of covalent binding of VPA-G and iso-VPA-G (using [¹⁴C]-labelled species) showed that adduct formation was much greater for iso-VPA-G. When [¹⁴C]-iso-VPA-G was reacted with MTP in the presence of sodium cyanide (to stabilize glycation adducts), subsequent separation into tubulin and MAPs fractions by ion exchange chromatography revealed that 78 and 22% of the covalent binding occurred with the MAPs and tubulin fractions respectively. These experiments support the notion of both covalent and reversible binding playing parts in the inhibition of microtubule formation from MTP (though the acyl glucuronide of VPA is less important than its rearrangement isomers in this regard), and that both tubulin and (perhaps more importantly) MAPs form adducts with acyl glucuronides.     

Focal loss of actin bundles causes microtubule redistribution and growth cone turning

It is commonly believed that growth cone turning during pathfinding is initiated by reorganization of actin filaments in response to guidance cues, which then affects microtubule structure to complete the turning process. However, a major unanswered question is how changes in actin cytoskeleton are induced by guidance cues and how these changes are then translated into microtubule rearrangement. Here, we report that local and specific disruption of actin bundles from the growth cone peripheral domain induced repulsive growth cone turning. Meanwhile, dynamic microtubules within the peripheral domain were oriented into areas where actin bundles remained and were lost from areas where actin bundles disappeared. This resulted in directional microtubule extension leading to axon bending and growth cone turning. In addition, this local actin bundle loss coincided with localized growth cone collapse, as well as asymmetrical lamellipodial protrusion. Our results provide direct evidence, for the first time, that regional actin bundle reorganization can steer the growth cone by coordinating actin reorganization with microtubule dynamics. This suggests that actin bundles can be potential targets of signaling pathways downstream of guidance cues, providing a mechanism for coupling changes in leading edge actin with microtubules at the central domain during turning.     

Movement of plasma-membrane-associated clathrin spots along the microtubule cytoskeleton

The current understanding of the role of plasma- membrane-associated clathrin suggests that clathrin-coated pits form at the sites of activated receptors and then, following internalization, the clathrin coat is rapidly shed. Utilizing total internal reflection fluorescence microscopy (TIR-FM), we have documented linear lateral motion of cell-surface-associated dsRed-clathrin spots parallel to the plasma membrane. Clathrin spot motility was observed in multiple cell lines (MDCK, CHO, Cos-7 and HeLa). In MDCK cells dsRed-clathrin spots moved along linear pathways up to 4 μm in length with rates of approximately 0.8 μm/s. Spots did not generally undergo internalization during movement. The motion of these puncta was coincident with the microtubule cytoskeleton, and depolymerization of microtubules reduced spot motility over 10-fold. Over-expression of the microtubule-associated protein tau-EGFP decreased spot run length by 40% without affecting the rate of movement. Thus dsRed-clathrin puncta move along the microtubule cytoskeleton parallel to the cell surface.     

Stabilization of the cyclin-dependent kinase 5 activator,p35, by paclitaxel decreases beta-amyloid toxicity in cortical neurons

One hallmark of Alzheimer's disease (AD) is the formation of neurofibrillary tangles, aggregated paired helical filaments composed of hyperphosphorylated tau. Amyloid-beta (Abeta) induces tau hyperphosphorylation, decreases microtubule (MT) stability and induces neuronal death. MT stabilizing agents have been proposed as potential therapeutics that may minimize Abeta toxicity and here we report that paclitaxel (taxol) prevents cell death induced by Abeta peptides, inhibits Abeta-induced activation of cyclin-dependent kinase 5 (cdk5) and decreases tau hyperphosphorylation. Taxol did not inhibit cdk5 directly but significantly blocked Abeta-induced calpain activation and decreased formation of the cdk5 activator, p25, from p35. Taxol specifically inhibited the Abeta-induced activation of the cytosolic cdk5-p25 complex, but not the membrane-associated cdk5-p35 complex. MT-stabilization was necessary for neuroprotection and inhibition of cdk5 but was not sufficient to prevent cell death induced by overexpression of p25. As taxol is not permeable to the blood-brain barrier, we assessed the potential of taxanes to attenuate Abeta toxicity in adult animals using a succinylated taxol analog (TX67) permeable to the blood-brain barrier. TX67, but not taxol, attenuated the magnitude of both basal and Abeta-induced cdk5 activation in acutely dissociated cortical cultures prepared from drug treated adult mice. These results suggest that MT-stabilizing agents may provide a therapeutic approach to decrease Abeta toxicity and neurofibrillary pathology in AD and other tauopathies.     

Phragmoplastin dynamics: multiple forms,microtubule association and their roles in cell plate formation in plants

We have characterized 4 of the 16 members of the family of dynamin-related proteins (DRP) in Arabidopsis. Three members, DRP1A (previously referred as ADL1), DRP1C and DRP1E, belong to the largest group of phragmoplastin-like proteins. DRP2A (ADL6) is one of the two members that contain a pleckstrin homology (PH) domain and a proline-rich (PR) motif, characteristics of animal dynamins. All four proteins interacted in yeast two-hybrid assays with phragmoplastin, and showed different patterns of localization at the forming cell plate during cytokinesis. GFP-tagged DRP1A and DRP1C proteins were found to be associated with the cytoskeleton in G1 phase of the cell cycle. The distribution pattern of DRP1A was sensitive to propyzamid and insensitive to cytochalasin D, suggesting that DRP1A is associated with microtubules and not actin filaments. The association of DRP1A with microtubules was confirmed in vitro by spin-down assays. A GTPase-defective phragmoplastin acted as a dominant negative mutant, reduced transport of vesicles to the cell plate and formed dense tubule-like structures in the cell plate. We propose that DRP1 proteins may provide an anchor for Golgi-derived vesicles to attach to microtubules, which in turn direct the vesicles to the forming cell plate during cytokinesis. Whereas the DRP1 subfamily members are involved in tubulization of membranes, DRP2 may be involved in endocytosis and membrane recycling via clathrin-coated vesicles.     

Role of microfilaments and microtubules in the invasion of INT-407 cells by Campylobacter jejuni

The internalization mechanisms triggered by Campylobacter jejuni were studied by invasion assays conducted with different inhibitors that act on the cytoskeleton structure of eukaryotic cells. The depolymerization of microfilaments by cytochalasin-D and that of microtubules by colchicines and nocodazole inhibited the uptake of C. jejuni into INT-407 cells in a dose-dependent manner. The inhibitory effect of microfilament depolymerization on C. jejuni internalization was more pronounced than that of microtubule depolymerization. By immunofluorescence microscopic observations, it was demonstrated that both microfilaments and microtubules were localized in INT-407 cells after C. jejuni infection. These data suggest that the internalization mechanism triggered by C. jejuni is associated with the combined effect of microfilaments and microtubules of host cells.     

Numerical simulations of microtubule self-organisation by reaction and diffusion

This article addresses the physical chemical processes underlying biological self-organisation by which a homogenous solution of reacting chemicals spontaneously self-organises. Theoreticians have predicted that self-organisation can arise from a coupling of reactive processes with molecular diffusion. In addition, the presence of an external field, such as gravity, at a critical moment early in the process may determine the morphology that subsequently develops. The formation, in-vitro, of microtubules, a constituent of the cellular skeleton, shows this type of behaviour. The preparations spontaneously self-organise by reaction-diffusion and the morphology that develops depends upon the presence of gravity at a critical bifurcation time early in the process. Here, we present numerical simulations of a population of microtubules that reproduce this behaviour. Microtubules can grow from one end whilst shrinking from the other. The shrinking end leaves behind a chemical trail of high tubulin concentration. Neighbouring microtubules preferentially grow into these regions, whilst avoiding regions of low tubulin concentration. The chemical trails produced by individual microtubules thus activate and inhibit the formation of neighbouring microtubules and this progressively leads to self-organisation. Gravity acts by way of its directional interaction with the macroscopic density fluctuations present in the solution arising from microtubule disassembly.     

Ran,a GTP-binding protein involved in nucleocytoplasmic transport and microtubule nucleation,relocates from the manchette to the centrosome region during rat spermiogenesis

Ran, a Ras-related GTPase, is required for transporting proteins in and out of the nucleus during interphase and for regulating the assembly of microtubules. cDNA cloning shows that rat testis, like mouse testis, expresses both somatic and testis-specific forms of Ran-GTPase. The presence of a homologous testis-specific form of Ran-GTPase in rodents implies that the Ran-GTPase pathway plays a significant role during sperm development. This suggestions is supported by distinct Ran-GTPase immunolocalization sites identified in developing spermatids. Confocal microscopy demonstrates that Ran-GTPase localizes in the nucleus of round spermatids and along the microtubules of the manchette in elongating spermatids. When the manchette disassembles, Ran-GTPase immunoreactivity is visualized in the centrosome region of maturing spermatids. The circumstantial observation that fractionated manchettes, containing copurified centrin-immunoreactive centrosomes, can organize a three-dimensional lattice in the presence of taxol and GTP, points to the role of Ran-GTPase and associated factors in microtubule nucleation as well as the potential nucleating function of spermatid centrosomes undergoing a reduction process. Electron microscopy demonstrates the presence in manchette preparations of spermatid centrosomes, recognized as such by their association with remnants of the implantation fossa, a dense plate observed only at the basal surface of developing spermatid and sperm nuclei. In addition, we have found importin beta1 immunoreactivity in the nucleus of elongating spermatids, a finding that, together with the presence of Ran-GTPase in the nucleus of round spermatids and the manchette, suggest a potential role of Ran-GTPase machinery in nucleocytoplasmic transport. Our expression and localization analysis, correlated with functional observations in other cell systems, suggest that Ran-GTPase may be involved in both nucleocytoplasmic transport and microtubules assembly, two critical events during the development of functional sperm. In addition, the manchette-to-centrosome Ran-GTPase relocation, together with the similar redistribution of various proteins associated to the manchette, suggest the existence of an intramanchette molecular transport mechanism, which may share molecular analogies with intraflagellar transport.