Sabina Winograd-Katz

Multiparametric analysis of focal adhesion formation by RNAi-mediated gene knockdown

Cell adhesion to the extracellular matrix is mediated by elaborate networks of multiprotein complexes consisting of adhesion receptors, cytoskeletal components, signaling molecules, and diverse adaptor proteins. To explore how specific molecular pathways function in the assembly of focal adhesions (FAs), we performed a high-throughput, high-resolution, microscopy-based screen. We used small interfering RNAs (siRNAs) to target human kinases, phosphatases, and migration- and adhesion-related genes. Multiparametric image analysis of control and of siRNA-treated cells revealed major correlations between distinct morphological FA features. Clustering analysis identified different gene families whose perturbation induced similar effects, some of which uncoupled the interfeature correlations. Based on these findings, we propose a model for the molecular hierarchy of FA formation, and tested its validity by dynamic analysis of FA formation and turnover. This study provides a comprehensive information resource on the molecular regulation of multiple cell adhesion features, and sheds light on signaling mechanisms regulating the formation of integrin adhesions.

Involvement of actin polymerization in podosome dynamics.

Podosomes, which are formed by different monocyte derivatives, are small adhesion structures whose coordinated dynamics and cytoskeletal reorganization drive their motile and invasive features. Using live-cell microscopy, we explored the temporal molecular steps of the de novo assembly and disassembly of podosomes in cultured osteoclasts. We demonstrate here that the earliest visible step in podosome assembly is the local accumulation of the plaque protein paxillin, along with cortactin, which stabilizes actin networks, followed by robust polymerization of actin filaments and their association with α-actinin. Only then is a local increase in integrin β3 levels apparent in the podosome ring domain. Thus, local actin polymerization in cortactin- and paxillin-rich locations nucleates podosome assembly before the local accumulation of β3 integrin. We further show that actin polymerization is also important for the recruitment and maintenance of plaque proteins in the mature podosome ring domain. Our model implies that core bundle dynamics play a central role in regulating podosome stability.

Recurrent inactivating RASA2 mutations in melanoma

Analysis of 501 melanoma exomes identified RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas. Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration. RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival. These findings identify RASA2 inactivation as a melanoma driver and highlight the importance of RasGAPs in cancer.

Analysis of the signaling pathways regulating Src-dependent remodeling of the actin cytoskeleton

Cell adhesion to the extracellular matrix is mediated by adhesion receptors, mainly integrins, which upon interaction with the extracellular matrix, bind to the actin cytoskeleton via their cytoplasmic domains. This association is mediated by a variety of scaffold and signaling proteins, which control the mechanical and signaling activities of the adhesion site. Upon transformation of fibroblasts with active forms of Src (e.g., v-Src), focal adhesions are disrupted, and transformed into dot-like contacts known as podosomes, and consisting of a central actin core surrounded by an adhesion ring. To clarify the mechanism underlying Src-dependent modulation of the adhesive phenotype, and its influence on podosome organization, we screened for the effect of siRNA-mediated knockdown of tyrosine kinases, MAP kinases and phosphatases on the reorganization of the adhesion-cytoskeleton complex, induced by a constitutively active Src mutant (SrcY527F). In this screen, we discovered several genes that are involved in Src-induced remodeling of the actin cytoskeleton. We further showed that knockdown of Src in osteoclasts abolishes the formation of the podosome-based rings and impairs cell spreading, without inducing stress fiber development. Our work points to several genes that are involved in this process, and sheds new light on the molecular plasticity of integrin adhesions.

Massive osteopetrosis caused by giant, non-functional osteoclasts in R51Q SNX10 mutant mice

In this study we report on the establishment and characterization of a novel knock-in mouse model that is homozygous for the R51Q mutation in the sorting nexin 10 (SNX10) protein. This mutation leads to massive, early-onset, and widespread osteopetrosis in the mutant mice, similar to that observed in humans who are homozygous for this mutation. The diseased mice exhibit multiple additional characteristics of the corresponding human osteopetrosis, including missing and impacted teeth, occasional osteomyelitis, stunted growth, failure to thrive, and a significantly-reduced lifespan. The phenotype of homozygous R51Q SNX10 osteoclasts is unique and defines a novel form of ARO that combines both lack of bone-resorbing activity and reduced cell numbers in vivo. Furthermore, mutant osteoclasts grown on bone develop a giant cell morphology, reaching sizes that are up to three orders of magnitude larger than osteoclasts from wild-type or heterozygous mice. These large osteoclasts display poor survival in vitro, which may account for their fewer numbers in vivo. Electron microscopy studies indicate that homozygous mutant osteoclasts exhibit severely impaired ruffled borders and are incapable of resorbing bone, providing a clear cellular basis for the osteopetrotic phenotype. We propose that the R51Q SNX10 mutation directly causes osteoporosis by affecting both osteoclast formation and function. We further conclude that the maximal size of osteoclasts is determined by an active and genetically-regulated mechanism in which SNX10 participates, and that it is disrupted by the R51Q SNX10 mutation.Summary The molecular mechanisms that regulate fusion of monocytes into functional osteoclasts are virtually unknown. We describe a knock-in mouse model for the R51Q mutation in sorting nexin 10 (SNX10) that exhibits osteopetrosis and related symptoms of patients of autosomal recessive osteopetrosis linked to this mutation. Osteopetrosis arises in homozygous R51Q SNX10 mice due to a unique combination of reduced numbers of osteoclasts that are non-functional. Fusion of mutant monocytes is deregulated and occurs rapidly and continuously to form giant, non-functional osteoclasts. Mutant osteoclasts mature quickly and survive poorly in vitro, possibly accounting for their scarcity in vivo. These cells also exhibit impaired ruffled borders, which are required for bone resorption, providing an additional basis for the osteopetrotic phenotype. More broadly, we propose that the maximal size of osteoclasts is actively determined by a genetically-regulated, cell-autonomous mechanism that limits precursor cell fusion, and for which SNX10 is required.

Image acquisition and understanding in high-throughput high-resolution cell-based screening applications

Quantitative interpretation of microscope images is more challenging the higher the resolution of the images is. The reward is rich multi-parametric characterization of subcellular structures and detailed description of cell responses to perturbations. This information is the basis of high- throughput cell-based screening, searching to discover new drugs and understand molecular mechanisms at the cell level. We have developed a fast screening microscope acquiring high-resolution images from cells cultured in plastic-bottom multi-well plates, [1-4] and are writing an automated pipeline for the analysis of Tera Bytes of images from high throughput screens. The platform includes database for storage and retrieval of images, visualization with easy linkage of the analyzed results to the original multi-color images, segmentation of objects in images (including cells, nuclei, cytoskeletal fibers and sub-cellular organelles), multi-parametric quantification of morphological and multicolor fluorescence intensities, and statistical comparisons to control wells displayed in color coded scores on the plate graphics. This system was successfully employed for screening of the effect of drugs, gene over-expression and siRNA of diverse cellular properties, including cell adhesion, migration, survival and cytoskeletal organization.