Ena/VASP Is Required for Neuritogenesis in the Developing Cortex

Kwiatkowski, Adam V.; Rubinson, Douglas A.; Dent, Erik W.; Veen, J. Edward Van; Leslie, Jonathan D.; Zhang, Jiangyang; Mebane, Leslie M.; Philippar, Ulrike; Pinheiro, Elaine M.; Burds, Aurora A.; Bronson, Roderick T.; Mori, Susumu; Fässler, Reinhard; Gertler, Frank B.

doi:10.1016/j.neuron.2007.09.008

Cited by 208 publications

(220 citation statements)

References 60 publications

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“…To determine if deletion of Ena/VASP proteins altered T-cell trafficking in vivo, we used EVL/VASP doubleknockout (dKO) mice (generously provided by Frank Gertler, Massachusetts Institute of Technology, Cambridge, MA) (31,32). We confirmed EVL and VASP deletion in T cells, and verified that Mena was not up-regulated as a compensatory mechanism in EVL/ VASP dKO T cells by Western blot analysis (Fig.…”

Section: Deletion Of Evl and Vasp Does Not Significantly Affect T-cellmentioning

confidence: 74%

Ena/VASP proteins regulate activated T-cell trafficking by promoting diapedesis during transendothelial migration

Estin

Thompson

Traxinger

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP-like (EVL) are cytoskeletal effector proteins implicated in regulating cell morphology, adhesion, and migration in various cell types. However, the role of these proteins in T-cell motility, adhesion, and in vivo trafficking remains poorly understood. This study identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naïve T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the impairment in α4 integrin (CD49d) expression and function. Unexpectedly, EVL/VASP dKO T cells did not exhibit alterations in shear-resistant adhesion to, or in crawling on, primary endothelial cells under physiologic shear forces. Instead, deletion of EVL and VASP impaired T-cell diapedesis. Furthermore, T-cell diapedesis became equivalent between control and EVL/VASP dKO T cells upon α4 integrin blockade. Overall, EVL and VASP selectively mediate activated T-cell trafficking by promoting the diapedesis step of transendothelial migration in a α4 integrin-dependent manner.A ctivated T-cell trafficking across the vascular endothelium is essential for ongoing immune surveillance of tissues and for effective immune responses to conditions such as infection and cancer. Conversely, in situations of immune dysregulation, inhibition of self-reactive T-cell trafficking represents a promising target for therapeutic immunomodulation. Disruption of these pathways, such as by antibody blockade of α4 integrins, is a highly effective approach to immunomodulation (1, 2). However, the molecular mechanisms by which chemokine receptor and adhesion molecule signaling induce the T-cell cytoskeletal machinery to promote extravasation are not yet fully elucidated.Transendothelial migration (TEM), the process by which T cells extravasate from the blood into tissues, is characterized by four distinct steps: rolling along the vascular wall, arrest or adhesion, intravascular crawling, and diapedesis across the endothelial barrier (3). Surface adhesion molecules play well-characterized roles in each step of the process. For example, the initial rolling step of TEM is facilitated by interactions between T-cell and endothelial selectins, whereas the adhesion, intravascular crawling, and diapedesis steps of TEM are mainly regulated by chemokineand shear force-stimulated modulation of lymphocyte functionassociated antigen 1 (LFA-1, αLβ2 integrin, CD11a/CD18) and very late antigen 4 (VLA-4, α4β1 integrin, CD49d/CD29) interactions with intracellular adhesion molecule 1 ...

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Section: Deletion Of Evl and Vasp Does Not Significantly Affect T-cellmentioning

confidence: 74%

Ena/VASP proteins regulate activated T-cell trafficking by promoting diapedesis during transendothelial migration

Estin

Thompson

Traxinger

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Primary hippocampal neurons and dorsal root ganglion (DRG) neurons cultured in the devices were obtained from embryonic day 14.5-16.5 mice as per protocols described in detail elsewhere 28,29,30 . Cells from micro-dissected cortices were dissociated and resuspended in serum-free media at a final concentration of 10 million cells/mL.…”

Section: Neuron Isolation and Culturementioning

confidence: 99%

A high-throughput microfluidic assay to study neurite response to growth factor gradients

et al. 2011

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Growth factor gradients have been implicated in many biological phenomena, including cell migration, neurite outgrowth and guidance in the nervous system. Current in vitro assays to study the effect of growth factors on neurite guidance are limited by their suitability for only substrate-bound gradients and 2D cultures. Here we describe a novel three-channel microfluidic device to study the role of chemogradients on neurite outgrowth and guidance in 3D scaffolds. Neurons packed in the bottom channel onto the collagen gel surface extended their neurites into the gel in three dimensions, and the growing neurites were exposed to a chemogradient orthogonal to the direction of their growth to quantify their response and turning. Experimental and computational studies showed that a linear stable chemogradient could be established in these devices within 30 min, and lasting for up to 48 h. Cell culture studies revealed the dramatic effect of chemoattractive (netrin-1, brain pulp) and chemorepulsive (slit-2) gradients on the migration and neurite guidance of hippocampal and dorsal root ganglion neurons cultured in these devices. The stable chemogradients in these 3-channel devices could not only be used to screen potential drugs suitable for neuron pathway regeneration under disease/ injury conditions, but also to study cancer cell migration and cell-cell interactions. AbstractStudying neurite guidance by diffusible or substrate bound gradients is challenging with current techniques. In this study, we present the design, fabrication and utility of a microfluidic device to study neurite guidance under chemogradients. Experimental and computational studies demonstrated the establishment of a steep gradient of guidance cue within 30 min and stable for up to 48 h. The gradient was found to be insensitive to external perturbations such as media change and movement of device. The effects of netrin-1 (0.1-10 µg/mL) and brain pulp (0.1 µL/mL) were evaluated for their chemoattractive potential on neurite turning, while slit-2 (62.5 or 250 ng/mL) was studied for its chemorepellant properties. Hippocampal or dorsal root ganglion (DRG) neurons were seeded into a micro-channel and packed onto the surface of a 3D collagen gel. Neurites grew into the matrix in three dimensions, and a gradient of guidance cue was created orthogonal to the direction of neurite growth to impact guidance. The average turning angle of each neurite was measured and averaged across multiple devices cultured under similar conditions to quantify the effect of guidance cue gradient. Significant positive turning towards gradient were measured in the presence of brain pulp and netrin-1 (1 µg/mL), relative to control cultures which received no external guidance cue (p < 0.001). Netrin-1 released from transfected fibroblasts had the most positive turning effect of all the chemoattractive cues tested (p

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“…As for the role of the actin cytoskeleton, although earlier work suggested that the actin depolymerizing drug cytochalasin D did not inhibit neurite formation in chick dorsal root ganglia neurons [40], more recent studies have demonstrated a critical role of the Ena/Mena/VASP family of proteins (abbreviated as Ena/VASP) in neurite formation [41,42]. Ena/VASP proteins regulate actin dynamics by antagonizing actin filament capping and promoting filament bundling [43,44].…”

Section: Introductionmentioning

confidence: 99%

Actin Aggregations Mark the Sites of Neurite Initiation

et al. 2016

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A salient feature of neurons is their intrinsic ability to grow and extend neurites, even in the absence of external cues. Compared to the later stages of neuronal development, such as neuronal polarization and dendrite morphogenesis, the early steps of neuritogenesis remain relatively unexplored. Here we showed that redistribution of cortical actin into large aggregates preceded neuritogenesis and determined the site of neurite initiation. Enhancing actin polymerization by jasplakinolide treatment effectively blocked actin redistribution and neurite initiation, while treatment with the actin depolymerizing agents latrunculin A or cytochalasin D accelerated neurite formation. Together, these results demonstrate a critical role of actin dynamics and reorganization in neurite initiation. Further experiments showed that microtubule dynamics and protein synthesis are not required for neurite initiation, but are required for later neurite stabilization. The redistribution of actin during early neuronal development was also observed in the cerebral cortex and hippocampus in vivo.

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Ena/VASP Is Required for Neuritogenesis in the Developing Cortex

Cited by 208 publications

References 60 publications

Ena/VASP proteins regulate activated T-cell trafficking by promoting diapedesis during transendothelial migration

Ena/VASP proteins regulate activated T-cell trafficking by promoting diapedesis during transendothelial migration

A high-throughput microfluidic assay to study neurite response to growth factor gradients

Actin Aggregations Mark the Sites of Neurite Initiation

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