Some of the most noticeable changes in the shape of a cell membrane are the formation of finger-like protrusions such as short filopodia (typically <5 μm long) that assist in environmental sensing and cell migration or tunneling nanotubes (TNTs) that connect cells up to 100 μm away for intercellular communication. Cell shape changes rely on the deformability of the plasma membrane, using actin polymerization to exert a pushing force against the membrane. Yet how actin is locally developed and what determines protrusion length scales has remained unknown. First through in vitro reconstitution experiments using purified proteins and giant vesicles we show that specific interactions between the curved, inverted Bin/Amphiphysin/Rvs (I-BAR) protein IRSp53 and phosphoinositides induce directional membrane-mediated interactions between I-BARs that facilitate protein clustering. This clustering served to recruit actin polymerases such as VASP (vasodilator-stimulated phosphoprotein) for actin growth locally at the membrane. Moreover, through micromanipulation by which tubes mimicking cell protrusions can be pulled from cells using laser tweezers, we assessed how IRSp53 enrichment coincides with actin growth and revealed that a critical activation step is necessary to precisely regulate the time and localization of protrusion growth. Secondly, the distance over which actin grows in these tubes was increased when branched networks reliant on Arp2/3 activity were inhibited pharmacologically with CK-666. Whole-cell proteomic analysis further revealed that upon Arp2/3 inhibition, proteins enhancing actin filament turnover and depolymerization were reduced and instead heightened interactions between IRSp53 and the actin filament bundling protein Eps8 (Epidermal growth factor receptor kinase substrate 8) were promoted. Altogether, this work demonstrates that IRSp53 is an efficient protrusion initiator, and once activated, IRSp53 readily triggers a positive feedback cascade for protrusion formation. Ultimately the length scales a protrusion achieves depends upon tuning protein-protein interactions and their involvement between branched and linear structures to favor actin filament assembly over disassembly.
Michael received his BS in Chemistry at the University of Central Oklahoma. For his graduate training in Physical Chemistry, he joined the group of Ka Yee C. Lee in the Department of Chemistry at The University Chicago where his thesis explored how membrane-mediated interactions regulate antimicrobial peptide (AMP) selectivity and their pore forming activity through a universal line tension reduction mechanism. After completing his PhD work in 2016, he moved to Paris, France to pursue his postdoctoral training and was a Marie Skłodowska-Curie and a Pasteur Foundation Fellowship recipient. Jointly split between two groups — the first headed by Patricia Bassereau in the Department of Physical Chemistry at Institut Curie, and the second headed by Chiara Zurzolo in the Department of Cell Biology and Infection at Istitut Pasteur — his research during that time addressed the formation of actin-based protrusions from short sensory filopodia to long tunneling nanotubes (TNTs) through the lens of curvature-sensitive protein assembly and force generation through actin filament polymerization. As of July 2023, he joined Bowdoin College as an Assistant Professor of Chemistry where his lab is developing biomimetic systems and micromanipulation measurements to continue addressing these themes in the context of neuronal cell function.