Proteins That Promote Filopodia Stability, but Not Number, Lead to More Axonal-Dendritic Contacts

Dendritic filopodia are dynamic protrusions that are thought to play an active role in synaptogenesis and serve as precursors to spine synapses. However, this hypothesis is largely based on a temporal correlation between filopodia formation and synaptogenesis. We investigated the role of filopodia in synapse formation by contrasting the roles of molecules that affect filopodia elaboration and motility, versus those that impact synapse induction and maturation. We used a filopodia inducing motif that is found in GAP-43, as a molecular tool, and found this palmitoylated motif enhanced filopodia number and motility, but reduced the probability of forming a stable axon-dendrite contact. Conversely, expression of neuroligin-1 (NLG-1), a synapse inducing cell adhesion molecule, resulted in a decrease in filopodia motility, but an increase in the number of stable axonal contacts. Moreover, RNAi knockdown of NLG-1 reduced the number of presynaptic contacts formed. Postsynaptic scaffolding proteins such as Shank1b, a protein that induces the maturation of spine synapses, increased the rate at which filopodia transformed into spines by stabilization of the initial contact with axons. Taken together, these results suggest that increased filopodia stability and not density, may be the rate-limiting step for synapse formation

Molecular Predictors of 3D Morphogenesis by Breast Cancer Cell Lines in 3D Culture

Correlative analysis of molecular markers with phenotypic signatures is the simplest model for hypothesis generation. In this paper, a panel of 24 breast cell lines was grown in 3D culture, their morphology was imaged through phase contrast microscopy, and computational methods were developed to segment and represent each colony at multiple dimensions. Subsequently, subpopulations from these morphological responses were identified through consensus clustering to reveal three clusters of round, grape-like, and stellate phenotypes. In some cases, cell lines with particular pathobiological phenotypes clustered together (e.g., ERBB2 amplified cell lines sharing the same morphometric properties as the grape-like phenotype). Next, associations with molecular features were realized through (i) differential analysis within each morphological cluster, and (ii) regression analysis across the entire panel of cell lines. In both cases, the dominant genes that are predictive of the morphological signatures were identified. Specifically, PPARγ has been associated with the invasive stellate morphological phenotype, which corresponds to triple-negative pathobiology. PPARγ has been validated through two supporting biological assays

Myosin-Va-interacting protein, RILPL2, controls cell shape and neuronal morphogenesis via Rac signaling

Neuronal morphology plays an essential role in neuronal function. The establishment and maintenance of neuronal morphology is intimately linked to the actin cytoskeleton; however, the molecular mechanisms that regulate changes in neuronal morphology are poorly understood. Here we identify a novel myosin-Va (MyoVa)-interacting protein, RILPL2, which regulates cellular morphology. Overexpression of this protein in young or mature hippocampal neurons results in an increase in the number of spine-like protrusions. By contrast, knockdown of endogenous RILPL2 in neurons by short hairpin RNA (shRNA) interference results in reduced spine-like protrusions, a phenotype rescued by overexpression of an shRNA-insensitive RILPL2 mutant, suggesting a role for RILPL2 in both the establishment and maintenance of dendritic spines. Interestingly, we demonstrate that RILPL2 and the Rho GTPase Rac1 form a complex, and that RILPL2 is able to induce activation of Rac1 and its target, p21-activated kinase (Pak). Notably, both RILPL2-mediated morphological changes and activation of Rac1-Pak signaling were blocked by expression of a truncated tail form of MyoVa or MyoVa shRNA, demonstrating that MyoVa is crucial for proper RILPL2 function. This might represent a novel mechanism linking RILPL2, the motor protein MyoVa and Rac1 with neuronal structure and function

Paralemmin-1 is expressed in lymphatic endothelial cells and modulates cell migration, cell maturation and tumor lymphangiogenesis

The lymphatic system, the network of lymphatic vessels and lymphoid organs, maintains the body fluid balance and ensures the immunological surveillance of the body. In the adult organism, the de novo formation of lymphatic vessels is mainly observed in pathological conditions. In contrast to the molecular mechanisms governing the generation of the lymphatic vasculature during embryogenesis, the processes underlying pathological lymphangiogenesis are less well understood. A genome-wide screen comparing the transcriptome of tumor-derived lymphatic endothelial cells with that of blood vessel endothelial cells identified paralemmin-1 as a protein prominently expressed in lymphatic endothelial cells. Paralemmin-1 is a lipid-anchored membrane protein that in fibroblasts and neurons plays a role in the regulation of cell shape, plasma membrane dynamics and cell motility. Here, we show that paralemmin-1 is expressed in tumor-derived lymphatic endothelial cells as well as in lymphatic endothelial cells of normal, non-tumorigenic tissue. Paralemmin-1 represses cell migration and delays the formation of tube-like structures of lymphatic endothelial cells in vitro by modulating cell-substrate adhesion, filopodia formation and plasma membrane blebbing. While constitutive genetic ablation of paralemmin-1 expression in mice has no effect on the development and physiological function of the lymphatic system, the loss of paralemmin-1 impaired tumor-associated lymphangiogenesis. Together, these results newly identify paralemmin-1 as a protein highly expressed in lymphatic endothelial cells. Similar to its function in neurons, it may link the cytoskeleton to the plasma membrane and thereby modulate lymphatic endothelial cell adhesion, migration and lymphangiogenesis