Annica K. B. Gad

RANTES promotes growth and survival of human first-trimester forebrain astrocytes

We have examined the role of α and β chemokines in the promotion of the ontogenetic development of the brain. RANTES was expressed preferentially in human fetal astrocytes in an age-dependent manner. Astrocytes from 5-week-old brains showed high proliferation and reduced survival, whereas 10-week-old astrocytes exhibited opposite effects. These effects were suppressed by anti-RANTES or anti-RANTES receptor antibodies and were enhanced by recombinant RANTES. RANTES induced tyrosine phosphorylation of several cellular proteins and nuclear translocation of STAT-1 in astrocytes. Interferon-γ (IFN-γ) was required for RANTES effects because RANTES induced IFN-γ and only 10-week-old astrocytes expressed the IFN-γ receptor. Blocking of IFN-γ with antibody reversed the effects of RANTES, indicating that cytokine/chemokine networks are critically involved in brain development.

Oncogenes induce a vimentin filament collapse mediated by HDAC6 that is linked to cell stiffness

Significance Oncogenes deregulate fundamental cellular functions, which can lead to the development of tumors and metastases. We show that oncogenes change the spatial organization of the vimentin fibers of the intracellular cytoskeleton, induce cell stiffness, and promote the invasive capacity of cells. We further show that this vimentin reorganization and increased cell stiffness requires histone deacetylase 6 (HDAC6). Taken together, these data support the concept that oncogenes can induce cellular stiffness via HDAC6-dependent reorganization of the vimentin filament network. These findings—that key molecules in oncogenic cell transformation, such as oncogenes and HDAC6, can modulate cell stiffness—highlight the importance of the need for further investigation of the mechanical properties of cells to better understand the mechanisms behind tumor and metastasis formation. Oncogenes deregulate fundamental cellular functions, which can lead to development of tumors, tumor-cell invasion, and metastasis. As the mechanical properties of cells govern cell motility, we hypothesized that oncogenes promote cell invasion by inducing cytoskeletal changes that increase cellular stiffness. We show that the oncogenes simian virus 40 large T antigen, c-Myc, and cyclin E induce spatial reorganization of the vimentin intermediate filament network in cells. At the cellular level, this reorganization manifests as increased width of vimentin fibers and the collapse of the vimentin network. At nanoscale resolution, the organization of vimentin fibers in these oncogene-expressing cells was more entangled, with increased width of the fibers compared with control cells. Expression of these oncogenes also resulted in up-regulation of the tubulin deacetylase histone deacetylase 6 (HDAC6) and altered spatial distribution of acetylated microtubules. This oncogene expression also induced increases in cellular stiffness and promoted the invasive capacity of the cells. Importantly, HDAC6 was required and sufficient for the structural collapse of the vimentin filament network, and was required for increased cellular stiffness of the oncogene-expressing cells. Taken together, these data are consistent with the possibility that oncogenes can induce cellular stiffness via an HDAC6-induced reorganization of the vimentin intermediate filament network.

The kinase-inhibitory domain of p21-activated kinase 1 (PAK1) inhibits cell cycle progression independent of PAK1 kinase activity

p21-activated kinase 1 (PAK1) is a mediator of downstream signaling from the small GTPases Rac and Cdc42. In its inactive state, PAK1 forms a homodimer where two kinases inhibit each other in trans. The kinase inhibitory domain (KID) of one molecule of PAK1 binds to the kinase domain of its counterpart and keeps it inactive. Therefore, the isolated KID of PAK1 has been widely used to specifically inhibit and study PAK function. Here, we show that the isolated KID induced a cell cycle arrest with accumulation of cells in the G1 phase of the cell cycle with an inhibition of cyclin D1 and D2 expression. This cell cycle arrest required the intact KID and was also induced by a mutated KID unable to block PAK1 kinase activity. Furthermore, the KID-induced cell cycle arrest could not be rescued by the expression of a constitutively active PAK1-T423E mutant, concluding that this arrest occurs independently of PAK1 kinase activity. Our results suggest that PAK1 through its KID inhibits cyclin D expression and thereby enforces a cell cycle arrest. Our results also call for serious precaution in the use of KID to study PAK function.

RhoD regulates cytoskeletal dynamics via the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules

RhoD has a role in actin dynamics that is distinct from RhoA, Rac1, and Cdc42. Data presented here indicate that RhoD binds the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules (WHAMM) and the related filamin A–binding protein FILIP1. WHAMM acts downstream of RhoD, and both proteins coordinate vital cellular processes, such as actin dynamics, cell attachment, and cell migration.

Oncogenic H-Ras V12 promotes anchorage-independent cytokinesis in human fibroblasts.

Cell anchorage is required for cell proliferation of untransformed cells, whereas anchorage-independent growth can be induced by oncogenes and is a hallmark of transformation. Whereas anchorage-dependent control of the progression of the G1 phase of the cell cycle has been extensively studied, it is less clear whether and how anchorage may control other cell cycle phases and whether oncogenes may affect such controls. Here, we found that lack of cell anchorage did not influence progression through the cell cycle S phase, G2 phase, or most of mitosis of primary human fibroblasts. However, unanchored fibroblasts could not complete cytokinesis. The cleavage furrow and central spindle were still formed in the absence of anchorage, but cells were unable to complete ingression, causing binucleation. Importantly, V12 H-Ras-transformed fibroblasts and two cancer cell lines progressed through the entire cell cycle without anchorage, including through cytokinesis. This indicates that oncogenic signaling may contribute to anchorage-independent growth and tumorigenesis by promoting the final cleavage furrow ingression during cytokinesis.

Rif proteins take to the RhoD: Rho GTPases at the crossroads of actin dynamics and membrane trafficking.

The Rif and RhoD proteins belong to the Rho subfamily of small GTPases. Rif and RhoD have for too long remained in the shadows of the better known Rho GTPases Cdc42, Rac1 and RhoA. With this review article, our aim is to provide the currently available information regarding Rif and RhoD. Taken together, the data available to date indicate that Rif and RhoD have unique roles in the regulation of actin dynamics, and that RhoD can link actin reorganisation to endosomal vesicle transport.

Majority of differentially expressed genes are down-regulated during malignant transformation in a four-stage model

The transformation of normal cells to malignant, metastatic tumor cells is a multistep process caused by the sequential acquirement of genetic changes. To identify these changes, we compared the transcriptomes and levels and distribution of proteins in a four-stage cell model of isogenically matched normal, immortalized, transformed, and metastatic human cells, using deep transcriptome sequencing and immunofluorescence microscopy. The data show that ∼6% (n = 1,357) of the human protein-coding genes are differentially expressed across the stages in the model. Interestingly, the majority of these genes are down-regulated, linking malignant transformation to dedifferentiation. The up-regulated genes are mainly components that control cellular proliferation, whereas the down-regulated genes consist of proteins exposed on or secreted from the cell surface. As many of the identified gene products control basic cellular functions that are defective in cancers, the data provide candidates for follow-up studies to investigate their functional roles in tumor formation. When we further compared the expression levels of four of the identified proteins in clinical cancer cohorts, similar differences were observed between benign and cancer cells, as in the cell model. This shows that this comprehensive demonstration of the molecular changes underlying malignant transformation is a relevant model to study the process of tumor formation.

Spatial organization of proteins in metastasizing cells

The ability of tumor cells to invade into the surrounding tissue is linked to defective adhesive and mechanical properties of the cells, which are regulated by cell surface adhesions and the intracellular filamentous cytoskeleton, respectively. With the aim to further reveal the underlying mechanisms and provide new strategies for early cancer diagnostics, we have used ultrahigh resolution stimulated emission depletion (STED) microscopy as a means to identify metastasizing cells, based on their subcellular protein distribution patterns reflecting their specific adhesive and mechanical properties. We have compared the spatial distribution of cell‐matrix adhesion sites and the vimentin filamentous systems in a matched pair of primary, normal, and metastatic human fibroblast cells. We found that the metastatic cells showed significantly increased densities and more homogenous distributions of nanoscale adhesion‐related particles. Moreover, they showed an increase in the number but reduced sizes of the areas of cell‐matrix adhesion complexes. The organization of the vimentin intermediate filaments was also found to be significantly different in the metastasizing cells, showing an increased entanglement and loss of directionality. Image analysis procedures were established, allowing an objective detection and characterization of these features and distinction of metastatic cells from their normal counterparts. In conclusion, our results suggest that STED microscopy provides a novel tool to identify metastasizing cells from a very sparse number of cells, based on the altered spatial distribution of the cell‐matrix adhesions and intermediate filaments.

Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions

The ability of cells to adhere and to exert contractile forces governs their capacity to move within an organism. The cytoskeletal regulators of the Rho GTPase proteins are involved in control of the contractile forces of cells. To elucidate the basis of cell migration, we analyzed contractile forces and nanoscale adhesion‐related particles in single cells expressing constitutively active variants of Rho GTPases by using traction‐force microscopy and ultra‐high‐resolution stimulated emission depletion microscopy, respectively. RhoAV14 induced large increases in the contractile forces of single cells, with Rac1L61 and RhoDV26 having more moderate effects. The RhoAV14‐ and RhoDV26‐induced forces showed similar spatial distributions and were accompanied by reduced or unaltered cell spreading. In contrast, the Rac1L61‐induced force had different, scattered, force distributions that were linked to increased cell spreading. All three of these Rho GTPase activities caused a loss of thick stress fibers and focal adhesions and a more homogenous distribution of nanoscale adhesion‐related particles over the ventral surface of the cells. Interestingly, only RhoAV14 increased the density of these particles. Our data suggest a Rac1‐specific mode for cells to generate contractile forces. Importantly, increased density and a more homogenous distribution of these small adhesion‐related particles promote cellular contractile forces.— Gad, A. K. B., Rönnlund, D., Spaar, A., Savchenko, A. A., Petranyi, G., Blom, H., Szekely, L., Widengren, J., Aspenström, P. Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions. FASEB J. 26, 2374‐2382 (2012). www.fasebj.org

Recombinant CD44-HABD is a novel and potent direct angiogenesis inhibitor enforcing endothelial cell-specific growth inhibition independently of hyaluronic acid binding

CD44 is the main cellular receptor for hyaluronic acid (HA). We previously found that overexpression of CD44 inhibited tumor growth of mouse fibrosarcoma cells in mice. Here, we show that soluble recombinant CD44 HA-binding domain (CD44-HABD) acts directly onto endothelial cells by inhibiting endothelial cell proliferation in a cell-specific manner. Consequently, soluble recombinant CD44-HABD also blocked angiogenesis in vivo in chick and mouse, and thereby inhibited tumor growth of various origins at very low doses (0.25 mg/kg × day). The antiangiogenic effect of CD44 is independent of its HA-binding capacity, since mutants deficient in HA binding still maintain their antiangiogenic and antiproliferative properties. Recombinant CD44-HABD represents a novel class of angiogenesis inhibitors based on a cell-surface receptor.