Thirumaran Thanabalu

A Bacillus sphaericus gene encoding a novel type of mosquitocidal toxin of 31.8 kDa

A size-fractionated genomic library of Bacillus sphaericus strain SSII-1 was constructed and screened for toxicity against larvae of the mosquito Culex quinquefasciatus (Cq). One toxin-producing clone, pS35, was identified and a 2.7-kb subclone was completely sequenced. An open reading frame of 879 bp encoding a 31.8-kDa protein (designated Mtx2) was identified. Purified, recombinant Mtx2 was toxic to Cq larvae. Mtx2 shows no significant homology to known insecticidal toxins, but has homology to two toxins active against mammalian cells, namely the epsilon-toxin of Clostridium perfringens and the cytotoxin of Pseudomonas aeruginosa. Thus, Mtx2 represents a new type of mosquitocidal toxin.

Hypoxia activated EGFR signaling induces epithelial to mesenchymal transition (EMT).

Metastasis is a multi-step process which requires the conversion of polarized epithelial cells to mesenchymal cells, Epithelial–Mesenchymal Transition (EMT). EMT is essential during embryonic morphogenesis and has been implicated in the progression of primary tumors towards metastasis. Hypoxia is known to induce EMT; however the molecular mechanism is still poorly understood. Using the A431 epithelial cancer cell line, we show that cells grown under hypoxic conditions migrated faster than cells grown under normal oxygen environment. Cells grown under hypoxia showed reduced adhesion to the extracellular matrix (ECM) probably due to reduced number of Vinculin patches. Growth under hypoxic conditions also led to down regulation of E-cadherin and up regulation of vimentin expression. The increased motility of cells grown under hypoxia could be due to redistribution of Rac1 to the plasma membrane as opposed to increased expression of Rac1. EGF (Epidermal Growth Factor) is a known inducer of EMT and growth of A431 cells in the absence of oxygen led to increased expression of EGFR (EGF Receptor). Treatment of A431 cells with EGF led to reduced cell adhesion to ECM, increased cell motility and other EMT characteristics. Furthermore, this transition was blocked by the monoclonal antibody Cetuximab. Cetuximab also blocked the hypoxia-induced EMT suggesting that cell growth under hypoxic conditions led to activation of EGFR signaling and induction of EMT phenotype.

Gene from tropical Bacillus sphaericus encoding a protease closely related to subtilisins from Antarctic bacilli.

We undertook to identify the protease(s) involved in the in vivo degradation of the 100 kDa mosquitocidal toxin (Mtx) from Bacillus sphaericus SSII-1 and isolated a B. sphaericus SSII-1 gene flanked upstream by a typical Shine-Dalgarno ribosome binding site and downstream by a strong rho-independent transcription terminator. The predicted ORF encodes a 432 amino acid protein with significant homology throughout its sequence to two subtilisin-like serine proteases from the Antarctic psychrophilic (cold-adapted) bacilli, TA39 and TA41. The predicted N-terminal sequence suggests that the B. sphaericus protease is related to sfericase, a partially characterized serine protease from B. sphaericus. Only B. sphaericus strains which produce Mtx-degrading protease activity harbour the subtilisin-like protease gene, suggesting that this protease may be responsible for or contribute to the degradation of Mtx in B. sphaericus SSII-1. A 36-kDa protease with Mtx-degrading activity and similar properties to sfericase was also purified from sporulated cultures of B. sphaericus SSII-1. Further studies are needed to determine the relationship of this protease to sfericase and to the predicted product of the subtilisin-like serine protease gene.

Functions of Vrp1p in cytokinesis and actin patches are distinct and neither requires a WH2/V domain

Vrp1 (verprolin, End5) is a Saccharomyces cerevisiae actin‐associated protein and is related to mammalian Wiskott–Aldrich syndrome protein (WASP)‐interacting protein (WIP). Vrp1‐deficient (vrp1Δ) cells are inviable at high temperature, have partially depolarized cortical actin patches and have defects in both actomyosin ring‐dependent and Hof1 (Cyk2)‐dependent pathways of cytokinesis. We demonstrate here that N‐Vrp11–364 and C‐Vrp1364–817 are each sufficient to restore viability, actomyosin ring constriction and Hof1 localization at 37°C to vrp1Δ. C‐Vrp1, like Vrp1, partially co‐localizes with cortical actin patches and restores actin patch polarization to vrp1Δ. Cortical localization of C‐Vrp1, but not Vrp1, requires Las17. N‐Vrp1 exhibits diffuse cytoplasmic localization and functions in cytokinesis without efficiently restoring polarization of cortical actin patches. N‐Vrp1 function is not abolished by mutations affecting the WASP homology 2 (WH2) [verprolin homology (V)] actin‐binding domain. N‐Vrp1 may function through the type I myosins and actin, while C‐Vrp1 may function through both Las17 (Bee1) and type I myosins. The functions of Vrp1 in viability at 37°C and cytokinesis do not require efficient localization to, and function in, the cortical actin cytoskeleton.

Verprolin function in endocytosis and actin organization - Roles of the Las17p (yeast WASP)-binding domain and a novel C-terminal actin-binding domain

Vrp1p (verprolin, End5p) is the yeast ortholog of human Wiskott–Aldrich syndrome protein (WASP)‐interacting protein (WIP). Vrp1p localizes to the cortical actin cytoskeleton, is necessary for its polarization to sites of growth and is also essential for endocytosis. At elevated temperature, Vrp1p becomes essential for growth. A C‐terminal Vrp1p fragment (C‐Vrp1p) retains the ability to localize to the cortical actin cytoskeleton and function in actin‐cytoskeleton polarization, endocytosis and growth. Here, we demonstrate that two submodules in C‐Vrp1p are required for actin‐cytoskeleton polarization: a novel C‐terminal actin‐binding submodule (CABS) that contains a novel G‐actin‐binding domain, which we call a verprolin homology 2 C‐terminal (VH2‐C) domain; and a second submodule comprising the Las17p‐binding domain (LBD) that binds Las17p (yeast WASP). The LBD localizes C‐Vrp1p to membranes and the cortical actin cytoskeleton. Intriguingly, the LBD is sufficient to restore endocytosis and growth at elevated temperature to Vrp1p‐deficient cells. The CABS also restores these functions, but only if modified by a lipid anchor to provide membrane association. Our findings highlight the role of Las17p binding for Vrp1p membrane association, suggest general membrane association may be more important than specific targeting to the cortical actin cytoskeleton for Vrp1p function in endocytosis and cell growth, and suggest that Vrp1p binding to individual effectors may alter their physiological activity.

Characterization of Wiskott–Aldrich syndrome (WAS) mutants using Saccharomyces cerevisiae

Wiskott-Aldrich syndrome (WAS) is caused by alterations in the WAS protein (WASP), and 80% of the missense mutations are located in the WH1 domain, the region essential for interaction with the WASP-interacting protein (WIP). It has been suggested that loss of WASP-WIP interaction is causal to the disease. Las17p (yeast WASP) is essential for growth at 37 degrees C. The growth defect of the las17Delta strain can be suppressed by the expression of human WASP together with WIP. Using the las17Delta strain, we have analyzed 52 missense mutations in the gene encoding WASP and found that 13 of these mutant proteins were unable to suppress the growth defect of the las17Delta strain. The majority of these 13 mutations cause the classic WAS in humans and are located within the WH1 domain, while none of the 12 mutations outside the WH1 domain abolished the activity of WASP in Saccharomyces cerevisiae cells. This suggests that some of the mutations (13 out of 40) in the WH1 domain cause the syndrome in humans by perturbing the WASP-WIP complex formation, while the rest of the mutations cause the syndrome without affecting the WASP-WIP complex formation, but may affect the activity of the complex.

The mammalian Verprolin, WIRE induces filopodia independent of N-WASP through IRSp53

The mammalian verprolin family of proteins, WIP (WASP Interacting Protein), CR16 (Corticoid Regulated) and WIRE (WIp-RElated) regulate the actin cytoskeleton through WASP/N-WASP (Wiskott Aldrich Syndrome Protein and Neural-WASP). In order to characterize the WASP/N-WASP-independent function of WIRE, we screened and identified IRSp53 (Insulin Receptor Substrate) as a WIRE interacting protein. Expression of IRSp53 with WIRE in N-WASP(-/-) mouse fibroblast cells induced filopodia while co-expression of IRSp53 with WIP did not. The induction of filopodia is dependent on WIRE-IRSp53 interaction as mutation in the SH3 domain of IRSp53 abolished WIRE-IRSp53 interaction as well as the ability to induce filopodia. Similarly, the Verprolin (V)-domain of WIRE is critical for IRSp53-WIRE interaction and for filopodia formation. The interaction between WIRE and IRSp53 is regulated by Cdc42 as mutations which abolish Cdc42-IRSp53 interaction lead to loss of IRSp53-WIRE interaction as shown by pull down assay. The plasma membrane localization of IRSp53 is dependent on Cdc42 and WIRE. Expression of Cdc42(G12V) (active mutant) with WIRE-IRSp53 caused significant increase in the number of filopodia per cell. Thus our results show that Cdc42 regulates the activity of IRSp53 by regulating the IRSp53-WIRE interaction as well as localization of the complex to plasma membrane to generate filopodia.

Insulin Receptor Substrate protein 53 kDa (IRSp53) is a negative regulator of myogenic differentiation

Fusion of mononucleated myoblasts to generate multinucleated myotubes is a critical step in skeletal muscle development. Filopodia, the actin cytoskeleton based membrane protrusions, have been observed early during myoblast fusion, indicating that they could play a direct role in myogenic differentiation. The control of filopodia formation in myoblasts remains poorly understood. Here we show that the expression of IRSp53 (Insulin Receptor Substrate protein 53kDa), a known regulator of filopodia formation, is down-regulated during differentiation of both mouse primary myoblasts and a mouse myoblast cell line C2C12. Over-expression of IRSp53 in C2C12 cells led to induction of filopodia and decrease in cell adhesion, concomitantly with inhibition of myogenic differentiation. In contrast, knocking down the IRSp53 expression in C2C12 cells led to a small but significant increase in myotube development. The decreased cell adhesion of C2C12 cells over-expressing IRSp53 is correlated with a reduction in the number of vinculin patches in these cells. Mutations in the conserved IMD domain (IRSp53 and MIM (missing in metastasis) homology domain) or SH3 domain of IRSp53 abolished the ability of this protein to inhibit myogenic differentiation and reduce cell adhesion. Over-expression of the IMD domain alone was sufficient to decrease the cell-extracellular matrix adhesion and to inhibit myogenesis in a manner dependent on its function in membrane shaping. Based on our data, we propose that IRSp53 is a negative regulator of myogenic differentiation which correlates with the observed down regulation of IRSp53 expression during myoblast differentiation to myotubes.

Verprolin: a cool set of actin-binding sites and some very HOT prolines.

Spatiotemporal organisation of eukaryotic cells is established and maintained by the cytoskeleton, a highly dynamic and complex network of structural and signalling proteins. Many components of the cytoskeleton are functionally and structurally conserved between humans and yeast. Among these are verprolin (Vrp1p) in yeast and its human ortholog Wiskott‐Aldrich syndrome protein (WASP)‐interacting protein (WIP). Much of our understanding of the function of these proteins has come from genetic analysis in yeast. Verprolin‐deficient yeast cells exhibit defects in cytokinesis, endocytosis, and actin cytoskeleton polarisation. Verprolin binds actin, the yeast ortholog of human WASP (Las17p or Bee1p), and the yeast ortholog of human PSTPIP1 (Hof1p or Cyk2p). We propose that verprolin acts as a chaperone that by transient bimolecular interactions maintains the proper function of its partners. Verprolin‐related proteins and partners are implicated in cancer, immunodeficiency, and neurodegeneration. Therefore, elucidating how verprolin functions will have major impacts in cell biology and medicine.

Vrp1p–Las17p interaction is critical for actin patch polarization but is not essential for growth or fluid phase endocytosis in S. cerevisiae

Vrp1p (yeast WIP) forms a protein complex with Las17p (yeast WASP), however the physiological significance of the interaction has not been fully characterized. Vrp1p residues, (788)MPKPR(792) are essential for Vrp1p-Las17p interaction. While C-Vrp1p(364-817) complements all the defects of the vrp1Δ strain, C-Vrp1p(364-817)(5A) ((788)AAAAA(792)) does not complement any of the defects, due to its inability to localize to cortical patches. Targeting C-Vrp1p(364-817)(5A) to membranes using CAAX motif (C-Vrp1p(364-817)(5A)-CAAX) rescued the growth and endocytosis defect but not the actin patch polarization defect of vrp1Δ. Vrp1p can localize to cortical patches, either by binding to Las17p through LBD (Las17 Binding Domain, Vrp1p(760-817)) or independent of Las17p through residues in N-Vrp1p(1-364). Unlike Vrp1p, Vrp1p(5A) localizes poorly to cortical patches and complements all the defects of vrp1Δ strain except actin patch polarization at elevated temperature. N-Vrp1p(1-364) complements all the defects of vrp1Δ strain except the actin patch polarization defect while N-Vrp1p(1-364)-LBD fusion protein complements all the defects. Thus our results show that while both Vrp1p and Las17p are essential for many cellular processes, the two proteins do not necessarily have to bind to each other to carry out these cellular functions. However, Las17p-Vrp1p interaction is essential for actin patch polarization at elevated temperature.