Spatiotemporal organization and mechanosensory function of podosomes

Podosomes are small, circular adhesions formed by cells such as osteoclasts, macrophages, dendritic cells, and endothelial cells. They comprise a protrusive actin core module and an adhesive ring module composed of integrins and cytoskeletal adaptor proteins such as vinculin and talin. Furthermore, podosomes are associated with an actin network and often organize into large clusters. Recent results from our laboratory and others have shed new light on podosome structure and dynamics, suggesting a revision of the classical “core-ring” model. Also, these studies demonstrate that the adhesive and protrusive module are functionally linked by the actin network likely facilitating mechanotransduction as well as providing feedback between these two modules. In this commentary, we briefly summarize these recent advances with respect to the knowledge on podosome structure and discuss force distribution mechanisms within podosomes and their emerging role in mechanotransduction.     

Spatiotemporal organization and mechanosensory function of podosomes

Podosomes are small, circular adhesions formed by cells such as osteoclasts, macrophages, dendritic cells, and endothelial cells. They comprise a protrusive actin core module and an adhesive ring module composed of integrins and cytoskeletal adaptor proteins such as vinculin and talin. Furthermore, podosomes are associated with an actin network and often organize into large clusters. Recent results from our laboratory and others have shed new light on podosome structure and dynamics, suggesting a revision of the classical “core-ring” model. Also, these studies demonstrate that the adhesive and protrusive module are functionally linked by the actin network likely facilitating mechanotransduction as well as providing feedback between these two modules. In this commentary, we briefly summarize these recent advances with respect to the knowledge on podosome structure and discuss force distribution mechanisms within podosomes and their emerging role in mechanotransduction.     

A functional link between dynamin and the actin cytoskeleton at podosomes

Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities for dynamin 2 and for the dynamin 2-binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and osteoclasts. Furthermore, GFP wild-type dynamin 2aa was targeted to podosomes. As shown by fluorescence recovery after photobleaching, GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aa(G273D) abolished podosomes while GFP-dynamin(K44A) was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin family and actin at attachment sites between cells and the substratum.     

Identification of amino acid residues lining the pore of a gap junction channel

Gap junctions represent a ubiquitous and integral part of multicellular organisms, providing the only conduit for direct exchange of nutrients, messengers and ions between neighboring cells. However, at the molecular level we have limited knowledge of their endogenous permeants and selectivity features. By probing the accessibility of systematically substituted cysteine residues to thiol blockers (a technique called SCAM), we have identified the pore-lining residues of a gap junction channel composed of Cx32. Analysis of 45 sites in perfused Xenopus oocyte pairs defined M3 as the major pore-lining helix, with M2 (open state) or M1 (closed state) also contributing to the wider cytoplasmic opening of the channel. Additional mapping of a close association between M3 and M4 allowed the helices of the low resolution map (Unger et al., 1999. Science. 283:1176-1180) to be tentatively assigned to the connexin transmembrane domains. Contrary to previous conceptions of the gap junction channel, the residues lining the pore are largely hydrophobic. This indicates that the selective permeabilities of this unique channel class may result from novel mechanisms, including complex van der Waals interactions of permeants with the pore wall, rather than mechanisms involving fixed charges or chelation chemistry as reported for other ion channels.     

Protein kinase C activation promotes microtubule advance in neuronal growth cones by increasing average microtubule growth lifetimes

We describe a novel mechanism for protein kinase C regulation of axonal microtubule invasion of growth cones. Activation of PKC by phorbol esters resulted in a rapid, robust advance of distal microtubules (MTs) into the F-actin rich peripheral domain of growth cones, where they are normally excluded. In contrast, inhibition of PKC activity by bisindolylmaleimide and related compounds had no perceptible effect on growth cone motility, but completely blocked phorbol ester effects. Significantly, MT advance occurred despite continued retrograde F-actin flow-a process that normally inhibits MT advance. Polymer assembly was necessary for PKC-mediated MT advance since it was highly sensitive to a range of antagonists at concentrations that specifically interfere with microtubule dynamics. Biochemical evidence is presented that PKC activation promotes formation of a highly dynamic MT pool. Direct assessment of microtubule dynamics and translocation using the fluorescent speckle microscopy microtubule marking technique indicates PKC activation results in a nearly twofold increase in the typical lifetime of a MT growth episode, accompanied by a 1.7-fold increase and twofold decrease in rescue and catastrophe frequencies, respectively. No significant effects on instantaneous microtubule growth, shortening, or sliding rates (in either anterograde or retrograde directions) were observed. MTs also spent a greater percentage of time undergoing retrograde transport after PKC activation, despite overall MT advance. These results suggest that regulation of MT assembly by PKC may be an important factor in determining neurite outgrowth and regrowth rates and may play a role in other cellular processes dependent on directed MT advance.     

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.     

Spatial organization of centrosome-attached and free microtubules in 3T3 fibroblasts

The spatial organization of microtubules is crucial for different cellular processes. It is traditionally supposed that fibroblasts have radial microtubule arrays consisting of long microtubules that run from the centrosome. However, a detailed analysis of the microtubule array in the internal cytoplasm has never been performed. In the current study, we used laser photobleaching to analyze the spatial organization of microtubules in the internal cytoplasm of cultured 3T3 fibroblasts. Cells were injected with Cy-3-labeled tubulin, after which the growth of microtubules in the centrosome region and peripheral parts of cytoplasm was assayed in the bleached zone. In most cases, microtubule growth in the bleached zone occurred rectilinearly; at distances of up to 5 μm, microtubules seldom bend more than 10°–15°. We considered a growing fragment of the microtubule as a vector with the beginning at the point of occurrence and the end at the point where the growth terminated (or the end point after 30 s if microtubule persistent growth proceeded for longer). We defined the direction of microtubule growth in different parts of the cell using these vectors and measured the angle of their deviation from the vector of comparison. In the area of the centrosome, we directed a comparison vector inside the bleached zone from the centrosome to the beginning of the growing microtubule segment; in the lamella and trailing part of the fibroblast, we used the vector of comparison directed along the long axis of the cell from its geometrical center to periphery. The microtubules growing straight away from the centrosome grew along the cell radius. However, at a distance of 10 μm from the centrosome, radially growing microtubules comprised 40% of the overall number, while at a distance of 20 μm, they made up only 25%. The rest of the microtubules grew in different directions, with the preferred angle between their growth direction and cell radius equaling around 90 °. In the lamella and trailing part of the fibroblast, 80% of all microtubules grew along the long axis of the cell or at an angle of no more than 20 °; 10–15% of microtubules grew along axis of the cell but towards the centrosome. Thus, in 3T3 fibroblasts, the radial system of microtubules is perturbed starting at a distance of several microns from the centrosome. In the internal cytoplasm, the microtubule system is completely disordered and, in the stretched parts of the polarized cell (lamella, trailing edge), the microtubule system again becomes well organized; microtubules are preferentially oriented along the long axis of the cell. From the results obtained, we conclude that the orderliness of microtubules at the periphery of the fibroblast is not a consequence of their growth from the centrosome; rather, their orientation is preset by local factors.     

Assembly and Function of the Actin Cytoskeleton of Yeast: Relationships between Cables and Patches

Actin in eukaryotic cells is found in different pools, with filaments being organized into a variety of supramolecular assemblies. To investigate the assembly and functional relationships between different parts of the actin cytoskeleton in one cell, we studied the morphology and dynamics of cables and patches in yeast. The fine structure of actin cables and the manner in which cables disassemble support a model in which cables are composed of a number of overlapping actin filaments. No evidence for intrinsic polarity of cables was found.To investigate to what extent different parts of the actin cytoskeleton depend on each other, we looked for relationships between cables and patches. Patches and cables were often associated, and their polarized distributions were highly correlated. Therefore, patches and cables do appear to depend on each other for assembly and function.Many cell types show rearrangements of the actin cytoskeleton, which can occur via assembly or movement of actin filaments. In our studies, dramatic changes in actin polarization did not include changes in filamentous actin. In addition, the concentration of actin patches was relatively constant as cells grew. Therefore, cells do not have bursts of activity in which new parts of the actin cytoskeleton are created.     

Vaccinia virus utilizes microtubules for movement to the cell surface

Vaccinia virus (VV) egress has been studied using confocal, video, and electron microscopy. Previously, intracellular-enveloped virus (IEV) particles were proposed to induce the polymerization of actin tails, which propel IEV particles to the cell surface. However, data presented support an alternative model in which microtubules transport virions to the cell surface and actin tails form beneath cell-associated enveloped virus (CEV) particles at the cell surface. Thus, VV is unique in using both microtubules and actin filaments for egress. The following data support this proposal. (a) Microscopy detected actin tails at the surface but not the center of cells. (b) VV mutants lacking the A33R, A34R, or A36R proteins are unable to induce actin tail formation but produce CEV and extracellular-enveloped virus. (c) CEV formation is inhibited by nocodazole but not cytochalasin D or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP1). (d) IEV particles tagged with the enhanced green fluorescent protein fused to the VV B5R protein moved inside cells at 60 microm/min. This movement was stop-start, was along defined pathways, and was inhibited reversibly by nocodazole. This velocity was 20-fold greater than VV movement on actin tails and consonant with the rate of movement of organelles along microtubules.     

Neurotrophin regulation of beta-actin mRNA and protein localization within growth cones.

Neurotrophins play an essential role in the regulation of actin-dependent changes in growth cone shape and motility. We have studied whether neurotrophin signaling can promote the localization of beta-actin mRNA and protein within growth cones. The regulated localization of specific mRNAs within neuronal processes and growth cones could provide a mechanism to modulate cytoskeletal composition and growth cone dynamics during neuronal development. We have previously shown that beta-actin mRNA is localized in granules that were distributed throughout processes and growth cones of cultured neurons. In this study, we demonstrate that the localization of beta-actin mRNA and protein to growth cones of forebrain neurons is stimulated by neurotrophin-3 (NT-3). A similar response was observed when neurons were exposed to forskolin or db-cAMP, suggesting an involvement of a cAMP signaling pathway. NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA. Localization of beta-actin mRNA was blocked by prior treatment of cells with Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A. Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of beta-actin mRNA. These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones.     

Association of the actin cytoskeleton with glass-adherent proteins in mouse peritoneal macrophages

Summary— When mouse peritoneal macrophages adherent to glass surface were removed by treatment with triethanolamine and Nonidet P-40, fine thread structures of unique loops were left behind on glass at the sites of cell adhesion. To examine the ultrastructural relationship between such looped threads and cytoskeletal components in glass-adherent macrophages, we successfully used the ‘zinc method’ to remove most of the cytoplasm including nuclei and to expose the cytoskeleton associated with the ventral plasma membrane. The cytoskeleton was seen to be mainly composed of actin filaments forming dense networks. The network contained scattered star-like foci from which actin filaments radiated. When the ventral plasma membrane-cytoskeleton complex was further treated with Nonidet P-40, the membrane was dissolved to expose the glass surface with actin foci persisting on glass. When the complex was removed by further treatment with Nonidet P-40 and DNase I, the looped threads became visible. Confocal laser microscopy of glass-adherent macrophages stained with fluorescent phalloidin showed the preferential distribution of F-actin in the ventral cytoplasm along the plasma membrane, where intense fluorescent spots were also scattered. Confocal interference reflection microscopy revealed densely populated dark dots and striae of focal contact, which corresponded in overall distribution to actin foci and looped threads. These observations suggest that actin cytoskeleton is closely associated with looped threads to reinforce cell adhesion to glass.     

Regulation of a formin complex by the microtubule plus end protein tea1p

The plus ends of microtubules have been speculated to regulate the actin cytoskeleton for the proper positioning of sites of cell polarization and cytokinesis. In the fission yeast Schizosaccharomyces pombe, interphase microtubules and the kelch repeat protein tea1p regulate polarized cell growth. Here, we show that tea1p is directly deposited at cell tips by microtubule plus ends. Tea1p associates in large "polarisome" complexes with bud6p and for3p, a formin that assembles actin cables. Tea1p also interacts in a separate complex with the CLIP-170 protein tip1p, a microtubule plus end-binding protein that anchors tea1p to the microtubule plus end. Localization experiments suggest that tea1p and bud6p regulate formin distribution and actin cable assembly. Although single mutants still polarize, for3Deltabud6Deltatea1Delta triple-mutant cells lack polarity, indicating that these proteins contribute overlapping functions in cell polarization. Thus, these experiments begin to elucidate how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth.     

An inactive pool of GSK-3 at the leading edge of growth cones is implicated in Semaphorin 3A signaling

Glycogen synthase kinase (GSK)-3 is a serine/threonine kinase that has been implicated in several aspects in embryonic development and several growth factor signaling cascades. We now report that an inactive phosphorylated pool of the enzyme colocalizes with F-actin in both neuronal and nonneuronal cells. Semaphorin 3A (Sema 3A), a molecule that inhibits axonal growth, activates GSK-3 at the leading edge of neuronal growth cones and in Sema 3A-responsive human breast cancer cells, suggesting that GSK-3 activity might play a role in coupling Sema 3A signaling to changes in cell motility. We show that three different GSK-3 antagonists (LiCl, SB-216763, and SB-415286) can inhibit the growth cone collapse response induced by Sema 3A. These studies reveal a novel compartmentalization of inactive GSK-3 in cells and demonstrate for the first time a requirement for GSK-3 activity in the Sema 3A signal transduction pathway.     

Unveiling the polarity of actin filaments by cryo-electron tomography

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Differential control of Eg5-dependent centrosome separation by Plk1 and Cdk1

Cyclin-dependent kinase 1 (Cdk1) is thought to trigger centrosome separation in late G2 phase by phosphorylating the motor protein Eg5 at Thr927. However, the precise control mechanism of centrosome separation remains to be understood. Here, we report that in G2 phase polo-like kinase 1 (Plk1) can trigger centrosome separation independently of Cdk1. We find that Plk1 is required for both C-Nap1 displacement and for Eg5 localization on the centrosome. Moreover, Cdk2 compensates for Cdk1, and phosphorylates Eg5 at Thr927. Nevertheless, Plk1-driven centrosome separation is slow and staggering, while Cdk1 triggers fast movement of the centrosomes. We find that actin-dependent Eg5-opposing forces slow down separation in G2 phase. Strikingly, actin depolymerization, as well as destabilization of interphase microtubules (MTs), is sufficient to remove this obstruction and to speed up Plk1-dependent separation. Conversely, MT stabilization in mitosis slows down Cdk1-dependent centrosome movement. Our findings implicate the modulation of MT stability in G2 and M phase as a regulatory element in the control of centrosome separation.     

Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals.     

Caldesmon affects actin organization at the leading edge and inhibits cell migration

The effect of the suppression of expression of the actin-binding protein caldesmon on the motility of nonmuscle cells has been studied. A more than a fivefold decrease in the content of this protein in cells by RNA interference led to the disturbance of the formation of actin stress fibers and acceleration of cell migration to the zone of injury of the monolayer. A stimulation of stationary cells by serum induced more than 1,5-fold accumulation of stress fibers only in control cells, but not in caldesmon-deficient cells. Similarly, the accumulation of actin filaments was observed in actively migrating cells of only wild type, but not in the cells with low caldesmon content. These changes occurred mainly at the leading edge of the migrating cell where the distinct structure of actin filaments was not seen in the absence of caldesmon. It was assumed that caldesmon inhibits cell migration due to the stabilization of actin in filaments and a decrease in the dynamics of monomeric actin at the leading edge of the migrating cell.     

Activity-Dependent Actin Remodeling at the Base of Dendritic Spines Promotes Microtubule Entry

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