Polarity establishment requires localized activation of Cdc42.

Establishment of cell polarity in animal and fungal cells involves localization of the conserved Rho-family guanosine triphosphatase, Cdc42, to the cortical region destined to become the "front" of the cell. The high local concentration of active Cdc42 promotes cytoskeletal polarization through various effectors. Cdc42 accumulation at the front is thought to involve positive feedback, and studies in the budding yeast Saccharomyces cerevisiae have suggested distinct positive feedback mechanisms. One class of mechanisms involves localized activation of Cdc42 at the front, whereas another class involves localized delivery of Cdc42 to the front. Here we show that Cdc42 activation must be localized for successful polarity establishment, supporting local activation rather than local delivery as the dominant mechanism in this system.

Inhibitory GEF phosphorylation provides negative feedback in the yeast polarity circuit.

Cell polarity is critical for the form and function of many cell types. During polarity establishment, cells define a cortical "front" that behaves differently from the rest of the cortex. The front accumulates high levels of the active form of a polarity-determining Rho-family GTPase (Cdc42, Rac, or Rop) that then orients cytoskeletal elements through various effectors to generate the polarized morphology appropriate to the particular cell type [1, 2]. GTPase accumulation is thought to involve positive feedback, such that active GTPase promotes further delivery and/or activation of more GTPase in its vicinity [3]. Recent studies suggest that once a front forms, the concentration of polarity factors at the front can increase and decrease periodically, first clustering the factors at the cortex and then dispersing them back to the cytoplasm [4-7]. Such oscillatory behavior implies the presence of negative feedback in the polarity circuit [8], but the mechanism of negative feedback was not known. Here we show that, in the budding yeast Saccharomyces cerevisiae, the catalytic activity of the Cdc42-directed GEF is inhibited by Cdc42-stimulated effector kinases, thus providing negative feedback. We further show that replacing the GEF with a phosphosite mutant GEF abolishes oscillations and leads to the accumulation of excess GTP-Cdc42 and other polarity factors at the front. These findings reveal a mechanism for negative feedback and suggest that the function of negative feedback via GEF inhibition is to buffer the level of Cdc42 at the polarity site.

CHC promotes tumor growth and angiogenesis through regulation of HIF-1α and VEGF signaling.

Pancreatic adenocarcinoma is an aggressive disease with a high mortality rate. In this study, we have newly generated a monoclonal antibody (mAb), Pa65-2, which specifically binds to pancreatic cancer cells and tumor blood vessels. The target protein of Pa65-2 is identified as human clathrin heavy chain (CHC). In vitro and In vivo study showed that suppression of CHC either by shRNA or by Pa65-2 inhibited tumor growth and angiogenesis. One of the key functions of CHC was to bind with the hypoxia-inducing factor (HIF)-1α protein, increasing the stability of this protein and facilitating its nuclear translocation, thereby regulating the expression of VEGF. Taken together, our findings indicate that CHC plays a role in the processes of tumorigenesis and angiogenesis. Pa65-2 antibody or CHC shRNA can potentially be used for pancreatic cancer therapy.

Cdc42p regulation of the yeast formin Bni1p mediated by the effector Gic2p.

Actin filaments are dynamically reorganized to accommodate ever-changing cellular needs for intracellular transport, morphogenesis, and migration. Formins, a major family of actin nucleators, are believed to function as direct effectors of Rho GTPases, such as the polarity regulator Cdc42p. However, the presence of extensive redundancy has made it difficult to assess the in vivo significance of the low-affinity Rho GTPase-formin interaction and specifically whether Cdc42p polarizes the actin cytoskeleton via direct formin binding. Here we exploit a synthetically rewired budding yeast strain to eliminate the redundancy, making regulation of the formin Bni1p by Cdc42p essential for viability. Surprisingly, we find that direct Cdc42p-Bni1p interaction is dispensable for Bni1p regulation. Alternative paths linking Cdc42p and Bni1p via "polarisome" components Spa2p and Bud6p are also collectively dispensable. We identify a novel regulatory input to Bni1p acting through the Cdc42p effector, Gic2p. This pathway is sufficient to localize Bni1p to the sites of Cdc42p action and promotes a polarized actin organization in both rewired and wild-type contexts. We suggest that an indirect mechanism linking Rho GTPases and formins via Rho effectors may provide finer spatiotemporal control for the formin-nucleated actin cytoskeleton.

Selective activation of SHP2 activity by cisplatin revealed by a novel chemical probe-based assay.

Src homology-2 (SH2) domain-containing phosphatase 2 (SHP2) is known to participate in several different signaling pathways to mediate cell growth, survival, migration, and differentiation. However, due to the lack of proper analytical tools, it is unclear whether the phosphatase activity of SHP2 is activated in most studies. We have previously developed an activity-based probe LCL2 that formed covalent linkage with catalytically active protein tyrosine phosphatases (PTPs). Here, by combining LCL2 with a SHP2 specific antibody, we established an assay system that enables the direct monitoring of SHP2 activity upon cisplatin treatment of cancer cells. The protocol is advantageous over conventional colorimetric or in-gel PTP assays as it is specific and does not require the use of radioisotope reagents. Using this assay, we found SHP2 activity was selectively activated by cisplatin. Moreover, the activation of SHP2 appeared to be specific for cisplatin as other DNA damage agents failed to activate the activity. Although the role of SHP2 activation by cisplatin treatments is still unclear to us, our results provide the first direct evidence for the activation of SHP2 during cisplatin treatments. More importantly, the concept of using activity-based probe in conjunction with target-specific antibodies could be extended to other enzyme classes.

The U3 small nucleolar ribonucleoprotein component Imp4p is a telomeric DNA-binding protein.

Imp4p is a component of U3 snoRNP (small nucleolar ribonucleoprotein) involved in the maturation of 18S rRNA. We have shown that Imp4p interacts with Cdc13p, a single-stranded telomere-binding protein involved in telomere maintenance. To understand the role of Imp4p in telomeres, we purified recombinant Imp4p protein and tested its binding activity towards telomeric DNA using electrophoretic mobility-shift assays. Our results showed that Imp4p bound specifically to single-stranded telomeric DNA in vitro. The interaction of Imp4p to telomeres in vivo was also demonstrated by chromatin immunoprecipitation experiments. Significantly, the binding of Imp4p to telomeres was not limited to yeast proteins, since the hImp4 (human Imp4) also bound to vertebrate single-stranded telomeric DNA. Thus we conclude that Imp4p is a novel telomeric DNA-binding protein that, in addition to its role in rRNA processing, might participate in telomere function.

Surfection: a new platform for transfected cell arrays.

Efficient high-throughput expression of genes in mammalian cells can facilitate large-scale functional genomic studies. Towards this aim, we developed a simple yet powerful method to deliver genes into cells by cationic polymers on the surface of substrates. Transfection can be achieved by directly contacting nucleic acid-cell mixtures with the cationic substrates, e.g. polyethylenimine/collagen-coated wells. This single-step matrix-surface- mediated transfection method, termed 'surfection', can efficiently deliver multiple plasmids into cells and can successfully assay siRNA-mediated gene silencing. This technology represents the easiest method to transfer combinations of genes in large-scale arrays, and is a versatile tool for live-cell imaging and cell-based drug screening.