Frank B. Gertler

A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.

RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses. But the resistance of important cell types to transduction by these approaches, both in vitro and in vivo, has limited the use of RNAi. Here we describe a lentiviral system for delivery of shRNAs into cycling and non-cycling mammalian cells, stem cells, zygotes and their differentiated progeny. We show that lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types and also in transgenic mice. Our lentiviral vectors should permit rapid and efficient analysis of gene function in primary human and animal cells and tissues and generation of animals that show reduced expression of specific genes. They may also provide new approaches for gene therapy.

Cytoskeletal dynamics and transport in growth cone motility and axon guidance

Recent studies indicate the actin and microtubule cytoskeletons are a final common target of many signaling cascades that influence the developing neuron. Regulation of polymer dynamics and transport are crucial for the proper growth cone motility. This review addresses how actin filaments, microtubules, and their associated proteins play crucial roles in growth cone motility, axon outgrowth, and guidance. We present a working model for cytoskeletal regulation of directed axon outgrowth. An important goal for the future will be to understand the coordinated response of the cytoskeleton to signaling cascades induced by guidance receptor activation.

Antagonism between Ena/VASP Proteins and Actin Filament Capping Regulates Fibroblast Motility

Cell motility requires lamellipodial protrusion, a process driven by actin polymerization. Ena/VASP proteins accumulate in protruding lamellipodia and promote the rapid actin-driven motility of the pathogen Listeria. In contrast, Ena/VASP negatively regulate cell translocation. To resolve this paradox, we analyzed the function of Ena/VASP during lamellipodial protrusion. Ena/VASP-deficient lamellipodia protruded slower but more persistently, consistent with their increased cell translocation rates. Actin networks in Ena/VASP-deficient lamellipodia contained shorter, more highly branched filaments compared to controls. Lamellipodia with excess Ena/VASP contained longer, less branched filaments. In vitro, Ena/VASP promoted actin filament elongation by interacting with barbed ends, shielding them from capping protein. We conclude that Ena/VASP regulates cell motility by controlling the geometry of actin filament networks within lamellipodia.

Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication

SCF ubiquitin ligases target phosphorylated substrates for ubiquitin-dependent proteolysis by means of adapter subunits called F-box proteins. The F-box protein Cdc4 captures phosphorylated forms of the cyclin-dependent kinase inhibitor Sic1 for ubiquitination in late G1 phase, an event necessary for the onset of DNA replication. The WD40 repeat domain of Cdc4 binds with high affinity to a consensus phosphopeptide motif (the Cdc4 phospho-degron, CPD), yet Sic1 itself has many sub-optimal CPD motifs that act in concert to mediate Cdc4 binding. The weak CPD sites in Sic1 establish a phosphorylation threshold that delays degradation in vivo, and thereby establishes a minimal G1 phase period needed to ensure proper DNA replication. Multisite phosphorylation may be a more general mechanism to set thresholds in regulated protein–protein interactions.

Mena is required for neurulation and commissure formation.

Mammalian enabled (Mena) is a member of a protein family thought to link signal transduction pathways to localized remodeling of the actin cytoskeleton. Mena binds directly to Profilin, an actin-binding protein that modulates actin polymerization. In primary neurons, Mena is concentrated at the tips of growth cone filopodia. Mena-deficient mice are viable; however, axons projecting from interhemispheric cortico-cortical neurons are misrouted in early neonates, and failed decussation of the corpus callosum as well as defects in the hippocampal commissure and the pontocerebellar pathway are evident in the adult. Mena-deficient mice that are heterozygous for a Profilin I deletion die in utero and display defects in neurulation, demonstrating an important functional role for Mena in regulation of the actin cytoskeleton.

A novel proline‐rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family

The ActA protein of the intracellular pathogen Listeria monocytogenes induces a dramatic reorganization of the actin‐based cytoskeleton. Two profilin binding proteins, VASP and Mena, are the only cellular proteins known so far to bind directly to ActA. This interaction is mediated by a conserved module, the EVH1 domain. We identify E/DFPPPPXD/E, a motif repeated 4‐fold within the primary sequence of ActA, as the core of the consensus ligand for EVH1 domains. This motif is also present and functional in at least two cellular proteins, zyxin and vinculin, which are in this respect major eukaryotic analogs of ActA. The functional importance of the novel protein–protein interaction was examined in the Listeria system. Removal of EVH1 binding sites on ActA reduces bacterial motility and strongly attenuates Listeria virulence. Taken together we demonstrate that ActA–EVH1 binding is a paradigm for a novel class of eukaryotic protein–protein interactions involving a proline‐rich ligand that is clearly different from those described for SH3 and WW/WWP domains. This class of interactions appears to be of general importance for processes dependent on rapid actin remodeling.

Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function

Entry of tumor cells into the blood stream is a critical step in cancer metastasis. Although significant progress has been made in visualizing tumor cell motility in vivo, the underlying mechanism of cancer cell intravasation remains largely unknown. We developed a microfluidic-based assay to recreate the tumor-vascular interface in three-dimensions, allowing for high resolution, real-time imaging, and precise quantification of endothelial barrier function. Studies are aimed at testing the hypothesis that carcinoma cell intravasation is regulated by biochemical factors from the interacting cells and cellular interactions with macrophages. We developed a method to measure spatially resolved endothelial permeability and show that signaling with macrophages via secretion of tumor necrosis factor alpha results in endothelial barrier impairment. Under these conditions intravasation rates were increased as validated with live imaging. To further investigate tumor-endothelial (TC-EC) signaling, we used highly invasive fibrosarcoma cells and quantified tumor cell migration dynamics and TC-EC interactions under control and perturbed (with tumor necrosis factor alpha) barrier conditions. We found that endothelial barrier impairment was associated with a higher number and faster dynamics of TC-EC interactions, in agreement with our carcinoma intravasation results. Taken together our results provide evidence that the endothelium poses a barrier to tumor cell intravasation that can be regulated by factors present in the tumor microenvironment.

The Disabled 1 Phosphotyrosine-Binding Domain Binds to the Internalization Signals of Transmembrane Glycoproteins and to Phospholipids

ABSTRACT Disabled gene products are important for nervous system development in drosophila and mammals. In mice, the Dab1 protein is thought to function downstream of the extracellular protein Reln during neuronal positioning. The structures of Dab proteins suggest that they mediate protein-protein or protein-membrane docking functions. Here we show that the amino-terminal phosphotyrosine-binding (PTB) domain of Dab1 binds to the transmembrane glycoproteins of the amyloid precursor protein (APP) and low-density lipoprotein receptor families and the cytoplasmic signaling protein Ship. Dab1 associates with the APP cytoplasmic domain in transfected cells and is coexpressed with APP in hippocampal neurons. Screening of a set of altered peptide sequences showed that the sequence GYXNPXY present in APP family members is an optimal binding sequence, with approximately 0.5 μM affinity. Unlike other PTB domains, the Dab1 PTB does not bind to tyrosine-phosphorylated peptide ligands. The PTB domain also binds specifically to phospholipid bilayers containing phosphatidylinositol 4P (PtdIns4P) or PtdIns4,5P2 in a manner that does not interfere with protein binding. We propose that the PTB domain permits Dab1 to bind specifically to transmembrane proteins containing an NPXY internalization signal.

Cables Links Cdk5 and c-Abl and Facilitates Cdk5 Tyrosine Phosphorylation, Kinase Upregulation, and Neurite Outgrowth

Cyclin-dependent kinase 5 (Cdk5) is a small serine/threonine kinase that plays a pivotal role during development of the CNS. Cables, a novel protein, interacts with Cdk5 in brain lysates. Cables also binds to and is a substrate of the c-Abl tyrosine kinase. Active c-Abl kinase leads to Cdk5 tyrosine phosphorylation, and this phosphorylation is enhanced by Cables. Phosphorylation of Cdk5 by c-Abl occurs on tyrosine 15 (Y15), which is stimulatory for p35/Cdk5 kinase activity. Expression of antisense Cables in primary cortical neurons inhibited neurite outgrowth. Furthermore, expression of active Abl resulted in lengthening of neurites. The data provide evidence for a Cables-mediated interplay between the Cdk5 and c-Abl signaling pathways in the developing nervous system.

An EMT–Driven Alternative Splicing Program Occurs in Human Breast Cancer and Modulates Cellular Phenotype

Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA–Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT–dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell–cell junction formation, and regulation of cell migration, were enriched among EMT–associated alternatively splicing events. Our analysis suggested that most EMT–associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT–associated splicing pattern. Expression of EMT–associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. The functional significance of EMT–associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT–associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression.