Abstract
Leading edge protrusion of migrating cells involves tightly coordinated changes in the plasma membrane and actin cytoskeleton. It remains unclear whether polymerizing actin filaments push and deform the membrane, or membrane deformation occurs independently and is subsequently stabilized by actin filaments. To address this question, we employed an ability of the membrane-binding I-BAR domain of IRSp53 to uncouple the membrane and actin dynamics and to induce filopodia in expressing cells. Using time-lapse imaging and electron microscopy of IRSp53-I-BAR-expressing B16F1 melanoma cells, we demonstrate that cells are not able to protrude or maintain durable long extensions without actin filaments in their interior, but I-BAR-dependent membrane deformation can create a small and transient space at filopodial tips that is subsequently filled with actin filaments. Moreover, the expressed I-BAR domain forms a submembranous coat that may structurally support these transient actin-free protrusions until they are further stabilized by the actin cytoskeleton. Actin filaments in the I-BAR-induced filopodia, in contrast to normal filopodia, do not have a uniform length, are less abundant, poorly bundled, and display erratic dynamics. Such unconventional structural organization and dynamics of actin in I-BAR-induced filopodia suggests that a typical bundle of parallel actin filaments is not necessary for generation and mechanical support of the highly asymmetric filopodial geometry. Together, our data suggest that actin filaments may not directly drive the protrusion, but only stabilize the space generated by the membrane deformation; yet, such stabilization is necessary for efficient protrusion.
Highlights
Leading edge protrusion of migrating cells is the first and essential step of the cell motility cycle, which is mediated by two types of actin-rich protrusive organelles, lamellipodia and filopodia
To understand how I-BAR-induced filopodia generate and maintain their shape, we investigated filopodia induced in B16F1 melanoma cells by the GFP-tagged I-BAR domain from IRSp53 (GFP-I-BAR)
A tight parallel bundle of long continuous actin filaments is believed to be necessary for mechanical stability and protrusion of filopodia and it is generally considered as a hallmark of highly elongated membrane protrusions [1]
Summary
Leading edge protrusion of migrating cells is the first and essential step of the cell motility cycle, which is mediated by two types of actin-rich protrusive organelles, lamellipodia and filopodia. Actin polymerization is clearly necessary for protrusion, there have been debates about whether growing actin filaments push the plasma membrane or they just fill the space created by other membrane-deforming mechanisms and stabilize the protrusion [2,3]. Answering this question has proven to be difficult, probably, because cells normally take special care of synchronizing these two processes. I-BARs may be able to form similar coats as they generate a striated pattern detectable by cryoelectron microscopy when assembled on giant unilamellar vesicles [13]
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