Cytoskeleton Regulation

Intermediate filaments (IF) are essential to maintain cellular and nuclear integrity and shape, to manage organelle distribution and motility, to control the trafficking and pH of intracellular vesicles, to prevent stress-induced cell death, and to support the correct distribution of specific proteins. Because of this, IF are likely to be targeted by a variety of pathogens, and may act in favor or against infection progress. As many IF functions remain to be identified, however, little is currently known about these interactions. Herpesviruses can infect a wide variety of cell types, and are thus bound to encounter the different types of IF expressed in each tissue. The analysis of these interrelationships can yield precious insights into how IF proteins work, and into how viruses have evolved to exploit these functions. These interactions, either known or potential, will be the focus of this review.

The intermediate filament protein, desmin, was purified from pork longissimus dorsi and incubated with either l-calpain, m-calpain or cathepsin B. Proteolysis of desmin was followed using SDS-PAGE and Western blotting. After incubation of desmin with the proteases, cleavage sites on the desmin molecule were identified by N-terminal sequencing of the different proteolytic fragments. Desmin incubated with either m-calpain or l-calpain was primarily cleaved in the head and tail region leaving the rod domain relatively intact even after prolonged incubation. Incubation with cathepsin B produces a sequential C-terminal degradation pattern characteristic of this dipeptylpeptidase. The substrate primary structure was not found to be essential for regulation of the proteolytic activity of the cysteine peptidases studied. However, the degradation patterns obtained imply that calpains are involved in degradation of desmin early post-mortem, targeting the non-helical region of the desmin molecule and resulting in depolymerisation and initial disorganisation of the intermediate filament structures of the muscle cell.

Out of the three main cytoskeletal fibers—actin filaments, intermediate filaments, and microtubules (MTs)—MTs have the largest diameter (outer diameter is ~25 nm). These microscopic hollow tubes are composed of the proteins α- and β-tubulin (γ-tubulin is localized to the centrosome and sometimes form caps on the minus ends of MTs) and are involved in a multitude of cellular activities and functions, including maintaining/carrying out/helping cell structural integrity, motility, division, organization, mechanotransduction, and intracellular transport. Other proteins that associate with MTs (MT-associated proteins (MAPs)) such as the motor proteins and MT-severing proteins regulate MTs and greatly increase the functionality of MTs.

Morphological changes are thought to contribute to the expression of long-term synaptic plasticity, a cellular basis for learning and memory. The mechanisms mediating the initiation and maintenance of the morphological changes are poorly understood. We repeatedly imaged the axonal arbors of mechanosensory neurons of Aplysia as they formed new synaptic varicosities and axonal branches after applications of serotonin that cause long-term synaptic facilitation. New varicosities formed exclusively from preexisting varicosities, by splitting or branch outgrowth. These changes were prevented by cytochalasin D, which blocks actin polymerization and the turnover of actin filaments. The suppression of the morphological changes by cytochalasin D did not impair their expression when cytochalasin D was removed 24 hr after exposure to serotonin. These results imply that serotonin induces persistent effects at preexisting presynaptic varicosities, which enhance actin polymerization, and that this is essential for presynaptic morphological changes of long-term facilitation.

Despite its decreasing frequency in developed countries, gastric cancer remains a significant health burden. The aim of the present study was to construct cDNA libraries and analyze differentially expressed genes related to this disease. Gene expression profiles were generated with suppressive subtraction hybridization (SSH). We constructed eight SSH libraries, four representing up-regulated genes and four representing down-regulated genes in tumor tissues. Our approach revealed that several genes are abnormally expressed in gastric cancer. We also identified global deregulation of several pathways involved in the maintenance of normal gastric homeostasis. The results of this study support the view that, as a result of complex pathogenesis, diversity of genomic aberrations and multiplicity of carcinogenic causes, gastric cancer cannot be reduced to a single molecule. Our results may contribute new insight into molecular aspects of the disease and may prove advantageous for future development of therapeutic targets and diagnostic molecular markers.

Morphological changes are thought to contribute to the expression of long-term synaptic plasticity, a cellular basis for learning and memory. The mechanisms mediating the initiation and maintenance of the morphological changes are poorly understood. We repeatedly imaged the axonal arbors of mechanosensory neurons of Aplysia as they formed new synaptic varicosities and axonal branches after applications of serotonin that cause long-term synaptic facilitation. New varicosities formed exclusively from preexisting varicosities, by splitting or branch outgrowth. These changes were prevented by cytochalasin D, which blocks actin polymerization and the turnover of actin filaments. The suppression of the morphological changes by cytochalasin D did not impair their expression when cytochalasin D was removed 24 hr after exposure to serotonin. These results imply that serotonin induces persistent effects at preexisting presynaptic varicosities, which enhance actin polymerization, and that this...

Intermediate filaments (IF) are one of the three main cytoskeletal filaments that have numerous functions stretching from maintaining structural integrity to helping form neuronal axons and constituting the nuclear lamina. Although they were initially named “intermediate” because of their width (~10 nm) compared to actin filaments (~7 nm) and wider myosin (~15 nm) filaments, they are now generally considered to be “intermediate” compared to actin filaments and microtubules (~25 nm). A variety of IFs are composed of numerous related proteins (unlike actin filaments and microtubules which are composed of only actin and tubulin proteins respectively) and they are generally categorized into 6 categories depending on the amino acid sequences and protein structures (classification can differ based on different classification methods). They are almost ubiquitously found in multicellular eukaryotic cells and are at least 10 times more abundant than actin filaments and microtubules.

Actin filaments assemble inside the nucleus in response to multiple cellular perturbations, including heat shock, protein misfolding, integrin engagement, and serum stimulation. We find that DNA damage also generates nuclear actin filaments—detectable by phalloidin and live-cell actin probes—with three characteristic morphologies: (i) long, nucleoplasmic filaments; (ii) short, nucleolus-associated filaments; and (iii) dense, nucleoplasmic clusters. This DNA damage-induced nuclear actin assembly requires two biologically and physically linked nucleation factors: Formin-2 and Spire-1/Spire-2. Formin-2 accumulates in the nucleus after DNA damage, and depletion of either Formin-2 or actin's nuclear import factor, importin-9, increases the number of DNA double-strand breaks (DSBs), linking nuclear actin filaments to efficient DSB clearance. Nuclear actin filaments are also required for nuclear oxidation induced by acute genotoxic stress. Our results reveal a previously unknown role for nuclear actin filaments in DNA repair and identify the molecular mechanisms creating these nuclear filaments.

During mitosis in budding yeast the nucleus first moves to the mother-bud neck and then into the neck. Both movements depend on interactions of cytoplasmic microtubules with the cortex. We investigated the mechanism of these movements in living cells using video analysis of GFP-labeled microtubules in wild-type cells and in EB1 and Arp1 mutants, which are defective in the first and second steps, respectively. We found that nuclear movement to the neck is largely mediated by the capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization. Efficient microtubule interactions with the capture site require that microtubules be sufficiently long and dynamic to probe the cortex. In contrast, spindle movement into the neck is mediated by microtubule sliding along the bud cortex, which requires dynein and dynactin. Free microtubules can also slide along the cortex of both bud and mother. Capture/shrinkage of microtubule ends also contributes to nuclear movement into the neck and can serve as a backup mechanism to move the nucleus into the neck when microtubule sliding is impaired. Conversely, microtubule sliding can move the nucleus into the neck even when capture/shrinkage is impaired.

Purpose: hMena (ENAH), a cytoskeleton regulatory protein involved in the regulation of cell motility and adhesion, is overexpressed in breast cancer. The aim of this study was to define at what stage of breast carcinogenesis hMena is overexpressed and to correlate hMena overexpression with established prognostic factors in breast cancer, focusing on human epidermal growth factor receptor-2 (HER-2). Experimental Design: hMena expression was assessed immunohistochemically in a prospective cohort of cases (n = 360) encompassing a highly representative spectrum of benign breast diseases associated with different risk of transformation, in situ, invasive, and metastatic tumors. Correlations with conventional pathologic and prognostic variables, such as proliferation index, hormonal receptor status, and HER-2 overexpression, were also evaluated. In vitro experiments were done to study the effect of neuregulin-1and Herceptin treatments on hMena expression. Results: hMena protein is undetectable in normal breast and is weakly expressed in a small percentage of low-risk benign diseases (9%), but displays a progressive and significant increase of positivity in benign lesions at higher risk of transformation (slightly increased risk 43%; moderate increased risk 67%), in in situ (72%), invasive (93%), and metastatic breast cancer (91%). A significant direct correlation with tumor size (P = 0.04), proliferation index (P < 0.0001), and HER-2 overexpression (P < 0.0001) and an inverse relationship with estrogen (P = 0.036) and progesterone receptors (P = 0.001) are found in invasive carcinomas. In vitro experiments show that neuregulin-1up-regulates, whereas Herceptin down-regulates, hMena expression. Conclusions: Our data provide new insights into the relevance of actin-binding proteins in human breast carcinogenesis and indicate hMena overexpression as a surrogate indicator in breast disease management.

The organization of cells into epithelium depends on cell interaction with both the extracellular matrix (ECM) and adjacent cells. The role of cell-cell adhesion in the regulation of epithelial topology is well-described. ECM is better known to promote cell migration and provide a structural scaffold for cell anchoring, but its contribution to multicellular morphogenesis is less well-understood. We developed a minimal model system to investigate how ECM affects the spatial organization of intercellular junctions. Fibronectin micropatterns were used to constrain the location of cell-ECM adhesion. We found that ECM affects the degree of stability of intercellular junction positioning and the magnitude of intra-and intercellular forces. Intercellular junctions were permanently displaced, and experienced large perpendicular tensional forces as long as they were positioned close to ECM. They remained stable solely in regions deprived of ECM, where they were submitted to lower tensional forces. The heterogeneity of the spatial organization of ECM induced anisotropic distribution of mechanical constraints in cells, which seemed to adapt their position to minimize both intra-and intercellular forces. These results uncover a morphogenetic role for ECM in the mechanical regulation of cells and intercellular junction positioning.

Assembled actin filaments support cellular signaling, intracellular trafficking, and cytokinesis. ATP hydrolysis triggered by actin assembly provides the structural cues for filament turnover in vivo. Here, we present the cryo-electron microscopic (cryo-EM) structure of filamentous actin (F-actin) in the presence of phosphate, with the visualization of some α-helical backbones and large side chains. A complete atomic model based on the EM map identified intermolecular interactions mediated by bound magnesium and phosphate ions. Comparison of the F-actin model with G-actin monomer crystal structures reveals a critical role for bending of the conserved proline-rich loop in triggering phosphate release following ATP hydrolysis. Crystal structures of G-actin show that mutations in this loop trap the catalytic site in two intermediate states of the ATPase cycle. The combined structural information allows us to propose a detailed molecular mechanism for the biochemical events, including actin polymerization and ATPase activation, critical for actin filament dynamics.► The cryo-EM structure of actin filament reveals intermolecular interactions ► Mg2+ and phosphate ions (Pi) play an important role in stabilizing the filament ► The bending of the Pro-rich loop triggers the activation of the ATPase activity ► A cavity in the catalytic site plays a role in releasing hydrolyzed γ-phosphates

Actin filaments are certainly believed to function as an intracellular signalling system; however, this is not confirmed by direct evidence. We used a two-layer actomyosin gel with a concentration gradient of the troponin-tropomyosin complex (TT-complex, Ca(2+)-sensitive system) between the two layers. To prepare one layer of the system, natural actomyosin (nAM) rich in TT-complex was used. To prepare the second layer, we used desensitized actomyosin (dAM) without the complex. All experimental studies were made in medium with a low ionic strength. Two phenomena were observed: (1) dAM blocks Ca(2+)-sensitivity of nAM when the dAM weight portion in the system (as well as in mixed nAM + dAM suspension) reaches 40% and more; further increase of the dAM portion does not affect the Ca(2+)-sensitivity; (2) it was electrophoretically shown that a rapid diffusion of the TT-complex from nAM gel into the dAM gel took place. The apparent diffusion coefficient for the TT-complex in dAM gel is ab...