Regulation of Actin Dynamics and Cellular Protrusions
Many of the ongoing projects in the lab involve the molecules that regulate actin dynamics. We have found that Ena/VASP proteins, a family of related molecules act as convergence points between signaling pathways and remodeling of the actin cytoskeleton. Ena/VASP proteins are required for a number of actin-based processes including axon guidance, cortical neuronal positioning, platelet aggregation, and fibroblast motility and epithelial sheet fusion and control of endothelial barrier function. Ena/VASP proteins also promote the actin driven motility of the bacterial pathogen Listeria monocytogenes.
Over the past few years, my laboratory has developed a mechanistic understanding for how Ena/VASP proteins control motility and formation of two types of cellular protrusions, lamellipodia and filopodia. Fibroblasts move faster in the absence of Ena/VASP activity. During normal motility, lamellipodia undergo cycles of extension and withdrawal. The net amount of protrusion over time correlates with the rate of cell movement.
Lamellipodia lacking Ena/VASP protrude slower than normal but continue to extend for much longer periods of time. Over time, this behavior of slow but persistent protrusion is integrated into higher net rates of overall cell movement. This led us to the surprising conclusion that persistence of individual lamellipodial protrusions is a more important parameter of whole cell speed than protrusive velocity of the lamellipodium.
Lamellipodial protrusion is driven by the formation of branched networks of actin filaments. Ena/VASP protein is normally concentrated at the distal tip of protruding lamellipodia. Lamellipodia lacking functional Ena/VASP proteins contain networks of abnormally short, highly branched filaments. Conversely, high levels of Ena/VASP in lamellipodia lead to the formation networks of long, sparsely branched filaments. These Ena/VASP-dependent alterations in actin geometry elicited the observed changes in lamellipodial dynamics when Ena/VASP proteins were absent or in excess.
Filopodia are comprised of parallel bundled actin filaments with their barbed ends directed outwards towards the plasma membrane. Ena/VASP proteins are concentrated at the tips of nascent and elongating filopodia. Ena/VASP function is required for efficient filopodia formation and elongation in fibroblasts as well as neurons. At a molecular level, Ena/VASP proteins interact directly with actin filaments at or near their barbed ends and shield them from capping proteins while permitting the filaments to continue to elongate. This biochemical activity correlates well with the observed functions of Ena/VASP in control of lamellipodial and filopodial dynamics. Ena/VASP anti-capping activity requires direct interaction with both actin filaments and monomer and is greatly enhanced by direct interaction with the actin-monomer binding protein profilin. Other studies ongoing in the lab continue to examine Ena/VASP anti-capping activity as well as how these molecules in turn interact with the signaling pathways regulating migration.
Interestingly, Ena/VASP proteins are substrates for the cyclic nucleotide-dependent kinases PKA and PKG. PKA phosphorylation is likely to be an important regulator of Ena/VASP function. Phosphorylation at a conserved amino-terminal PKA site correlates with Ena/VASP-dependent filopodia formation in neurons. Since PKA and PKG are thought to regulate growth cone response to a number of attractive and repulsive guidance factors, we speculate that Ena/VASP proteins play a key role in such responses. Currently we are analyzing how different phosphorylation sites stimulate or inhibit Ena/VASP activity.
We also identified lamellipodin (Lpd), an Ena/VASP-binding protein concentrated in lamellipodial and filopodial tips. Interestingly, Lpd associates directly with PI(3,4)P2, a phosphoinositide enriched in the leading edges of cells responding to chemotactic signals and with proteins of the RAS GTPase superfamily. RNAi depletion of Lpd results in a near complete failure of lamellipodial protrusion and concomitant reduction in F-actin levels, a phenotype more severe than the one observed in Ena/VASP-deficient cells. Therefore, we hypothesize that Lpd link signaling by RAS GTPases and phospholipids to actin dynamics via mechanisms that include Ena/VASP proteins and other actin regulatory molecules. We are using proteomics and other approaches to identify other potential signaling and actin regulatory proteins that are in complex with Ena/VASP and Lpd within lamellipodia and filopodia. We have also generated Lpd knockout mice and expect to begin phenotypic analysis of the mutants by early 2006.
Regulation of Axon formation and Guidance
Another form of actin-based motility that we study involves neuronal growth cone migration in response to guidance cues. Growth cone movement is dominated by filopodia, structures formed by parallel bundles of actin filaments. The lamellipodial veil in between growth cone filopodia is usually concave, unlike the convex shape of fibroblast lamellipodia. The concave shape suggests that growth cone “lamellipodia” may not be a protrusive structure in the traditional sense, but may be dragged along by the motile forces of filopodia. In contrast to the highly branched arrays within fibroblast lamellipodia, the actin network in growth cones is dominated by thick parallel bundles of filaments that comprise filopodia with a less dense network of relatively long filaments and a comparatively low density of branched networks near the edge of the lamellipodial veil. We hypothesize that growth cones and fibroblasts utilize different balances of actin modifying proteins and structures during movement and guidance and are testing this hypothesis by a variety of approaches.
Recently, we generated mutant mouse embryos that lack all three Ena/VASP proteins. Triple mutants survive until late embryogenesis and exhibit a number of interesting phenotypes. Mutant cortices exhibit an almost complete loss of axonal fibers. Interestingly, some neurons migrate beyond the outer layer of the cortex to form ectopic clusters. Such ectopia form axons as do other types of neurons in the mutants including retinal neurons and neurons that form spinal nerves. At a cellular level, loss of Ena/VASP results in almost complete absence of filopodia on neurons. We hypothesize that filopodia serve to guide and stabilize microtubules at the cell periphery and that microtubule invasion is a key step in axonogenesis. We are using the triple mutant animals to study axonogenesis and to explore the function of Ena/VASP in response to axon guidance cues.
The spread of cancer from a primary tumor to secondary sites requires tumor cells to invade adjacent tissues and migrate towards blood vessels. Working with our collaborator, John Condeelis (Albert Einstein College of Medicine), we have found that Ena/VASP proteins are upregulated during breast cancer invasion in vivo. Ena/VASP proteins are concentrated in invadopodia, specialized matrix-secreting protrusions found in invasive cells. We are currently examining the role of Ena/VASP in tumor cell invasion in vitro and in vivo. Interestingly, we have identified new Ena/VASP interacting proteins that are also concentrated in invadopodia.
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Menzies AS, Aszodi A, Williams SE, Pfeifer A, Wehman AM, Goh KL, Mason CA, Fassler R, Gertler FB. Mena and vasodilator-stimulated phosphoprotein are required for multiple actin-dependent processes that shape the vertebrate nervous system. J Neurosci. 24:8029-38 (2004).
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Strasser GA, Rahim NA, VanderWaal KE, Gertler FB, Lanier LM. Arp2/3 is a negative regulator of growth cone translocation. Neuron. 43:81-94 (2004).
Mejillano MR, Kojima S, Applewhite DA, Gertler FB, Svitkina TM, Borisy GG. Lamellipodial versus filopodial mode of the actin nanomachinery: pivotal role of the filament barbed end. Cell. 118:363-73 (2004).
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