Guiding Irregular Nuclear Morphology on Nanopillar Arrays for Malignancy Differentiation in Tumor Cells.

For more than a century, abnormal nuclei in tumor cells, presenting subnuclear invaginations and folds on the nuclear envelope, have been known to be associated with high malignancy and poor prognosis. However, current nuclear morphology analysis focuses on the features of the entire nucleus, overlooking the malignancy-related subnuclear features in nanometer scale. The main technical challenge is to probe such tiny and randomly distributed features inside cells. We here employ nanopillar arrays to guide subnuclear features into ordered patterns, enabling their quantification as a strong indicator of cell malignancy. Both breast and liver cancer cells were validated as well as the quantification of nuclear abnormality heterogeneity. The alterations of subnuclear patterns were also explored as effective readouts for drug treatment. We envision that this nanopillar-enabled quantification of subnuclear abnormal features in tumor cells opens a new angle in characterizing malignant cells and studying the unique nuclear biology in cancer.

Fibroblast rejuvenation by mechanical reprogramming and redifferentiation.

Over the course of the aging process, fibroblasts lose contractility, leading to reduced connective-tissue stiffness. A promising therapeutic avenue for functional rejuvenation of connective tissue is reprogrammed fibroblast replacement, although major hurdles still remain. Toward this, we recently demonstrated that the laterally confined growth of fibroblasts on micropatterned substrates induces stem-cell-like spheroids. In this study, we embedded these partially reprogrammed spheroids in collagen-I matrices of varying densities, mimicking different three-dimensional (3D) tissue constraints. In response to such matrix constraints, these spheroids regained their fibroblastic properties and sprouted to form 3D connective-tissue networks. Interestingly, we found that these differentiated fibroblasts exhibit reduced DNA damage, enhanced cytoskeletal gene expression, and actomyosin contractility. In addition, the rejuvenated fibroblasts show increased matrix protein (fibronectin and laminin) deposition and collagen remodeling compared to the parental fibroblast tissue network. Furthermore, we show that the partially reprogrammed cells have comparatively open chromatin compaction states and may be more poised to redifferentiate into contractile fibroblasts in 3D-collagen matrix. Collectively, our results highlight efficient fibroblast rejuvenation through laterally confined reprogramming, which has important implications in regenerative medicine.

Cell geometry dictates TNFα-induced genome response.

Cells in physiology integrate local soluble and mechanical signals to regulate genomic programs. Whereas the individual roles of these signals are well studied, the cellular responses to the combined chemical and physical signals are less explored. Here, we investigated the cross-talk between cellular geometry and TNFα signaling. We stabilized NIH 3T3 fibroblasts into rectangular anisotropic or circular isotropic geometries and stimulated them with TNFα and analyzed nuclear translocation of transcription regulators -NFκB (p65) and MKL and downstream gene-expression patterns. We found that TNFα induces geometry-dependent actin depolymerization, which enhances IκB degradation, p65 nuclear translocation, nuclear exit of MKL, and sequestration of p65 at the RNA-polymerase-II foci. Further, global transcription profile of cells under matrix-TNFα interplay reveals a geometry-dependent gene-expression pattern. At a functional level, we find cell geometry affects TNFα-induced cell proliferation. Our results provide compelling evidence that fibroblasts, depending on their geometries, elicit distinct cellular responses for the same cytokine.

The tyrosine phosphatase TC48 interacts with and inactivates the oncogenic fusion protein BCR-Abl but not cellular Abl.

The chimeric oncoprotein BCR-Abl exhibits deregulated protein tyrosine kinase activity and is responsible for the pathogenesis of certain human leukemias, such as chronic myelogenous leukemia. The activities of cellular Abl (c-Abl) and BCR-Abl are stringently regulated and the cellular mechanisms involved in their inactivation are poorly understood. Protein tyrosine phosphatases can negatively regulate Abl mediated signaling by dephosphorylating the kinase and/or its substrates. This study investigated the ability of the intracellular T cell protein tyrosine phosphatase (TCPTP/PTPN2) to dephosphorylate and regulate the functions of BCR-Abl and c-Abl. TCPTP is expressed as two alternately spliced isoforms - TC48 and TC45, which differ in their C-termini and localize to the cytoplasm and nucleus respectively. We show that TC48 dephosphorylates BCR-Abl but not c-Abl and inhibits its activity towards its substrate, CrkII. Y1127 and Y1294 residues whose phosphorylation corresponds with BCR-Abl activation status were the primary sites targeted by TC48. Co-localization and immunoprecipitation experiments showed that TC48 interacted with BCR-Abl but not with c-Abl, and BCR domain was sufficient for interaction. TC48 expression resulted in the stabilization of Bcr-Abl protein dependent on its phosphatase activity. Inactivation of cellular TC48 in K562 cells by stable expression of a dominant negative catalytically inactive mutant TC48, enhanced proliferation. TC48 expressing K562 clones showed reduced proliferation and enhanced sensitivity to STI571 compared to control clones suggesting that TC48 can repress the growth of CML cells. This study identifies a novel cellular regulator that specifically inhibits the activity of oncogenic BCR-Abl but not that of the cellular Abl kinase.

TC-PTP dephosphorylates the guanine nucleotide exchange factor C3G (RapGEF1) and negatively regulates differentiation of human neuroblastoma cells.

The guanine nucleotide exchange factor, C3G (RapGEF1), functions in multiple signaling pathways involved in cell adhesion, proliferation, apoptosis and actin reorganization. C3G is regulated by tyrosine phosphorylation on Y504, known to be mediated by c-Abl and Src family kinases. In the present study we explored the possibility of cellular phospho-C3G (pC3G) being a substrate of the intracellular T-cell protein tyrosine phosphatase TC-PTP (PTPN2) using the human neuroblastoma cell line, IMR-32. In vivo and in vitro binding assays demonstrated interaction between C3G and TC-PTP. Interaction is mediated through the Crk-binding region of C3G and C-terminal noncatalytic residues of TC-PTP. C3G interacted better with a substrate trap mutant of TC48 and this complex formation was inhibited by vanadate. Endogenous pC3G colocalized with catalytically inactive mutant TC48 in the Golgi. Expression of TC48 abrogated pervanadate and c-Src induced phosphorylation of C3G without affecting total cellular phospho-tyrosine. Insulin-like growth factor treatment of c-Src expressing cells resulted in dephosphorylation of C3G dependent on the activity of endogenous TC48. TC48 expression inhibited forskolin induced tyrosine phosphorylation of C3G and neurite outgrowth in IMR-32 cells. Our results identify a novel Golgi localized substrate of TC48 and delineate a role for TC48 in dephosphorylation of substrates required during differentiation of human neuroblastoma cells.

Signalling to actin: role of C3G, a multitasking guanine-nucleotide-exchange factor.

C3G (Crk SH3-domain-binding guanine-nucleotide-releasing factor) is a ubiquitously expressed member of a class of molecules called GEFs (guanine-nucleotide-exchange factor) that activate small GTPases and is involved in pathways triggered by a variety of signals. It is essential for mammalian embryonic development and many cellular functions in adult tissues. C3G participates in regulating functions that require cytoskeletal remodelling such as adhesion, migration, maintenance of cell junctions, neurite growth and vesicle traffic. C3G is spatially and temporally regulated to act on Ras family GTPases Rap1, Rap2, R-Ras, TC21 and Rho family member TC10. Increased C3G protein levels are associated with differentiation of various cell types, indicating an important role for C3G in cellular differentiation. In signalling pathways, C3G serves functions dependent on catalytic activity as well as protein interaction and can therefore integrate signals necessary for the execution of more than one cellular function. This review summarizes our current knowledge of the biology of C3G with emphasis on its role as a transducer of signals to the actin cytoskeleton. Deregulated C3G may also contribute to pathogenesis of human disorders and therefore could be a potential therapeutic target.

C3G is required for c-Abl-induced filopodia and its overexpression promotes filopodia formation.

The Rap1 guanine nucleotide exchange factor, C3G (also known as Rap1GEF-1) is involved in signaling from growth factors, cytokines and integrins and plays a role in cell adhesion and migration, but the mechanism by which C3G regulates various cellular functions is poorly understood. We, therefore, investigated the ability of C3G to affect actin cytoskeleton-dependent morphological changes in cells. Using RNA interference, we provide evidence that C3G is required for c-Abl-induced filopodia during cell spreading on fibronectin. C3G expression induces actin cytoskeletal reorganization and promotes filopodia formation independent of its catalytic activity. It showed enrichment at filopodia tips characteristic of molecules involved in filopodia dynamics. C3G-induced filopodia were not inhibited by dominant negative mutants of Rho, Rac and Cdc42, but required Abl catalytic activity. Coexpression of N-Wasp-Crib inhibited C3G induced as well as c-Abl-induced filopodia and wiskostatin, a pharmacological inhibitor of N-Wasp attenuates C3G-induced filopodia. Cellular C3G interacts with c-Abl and C3G expression results in enhanced localization of endogenous c-Abl in the cytoplasm. We suggest that C3G and c-Abl function in an interdependent manner, in linking external signals to remodeling the cytoskeleton to induce filopodia.