Thesis manuscript: Stabilization and motility mechanism of blebs in cancer cells
Creators: name: ARCOS, Juan Manuel GARCIA
Publisher: name: figshare
Year: 2021
Subjects: subject: 111201 Cancer Cell Biology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Cell Biology
subject: Biophysics

description: Previously associated with apoptosis, blebs have arisen in the past decade as <b>important structures for amoeboid cell migration</b>, particularly for cancer cells. Blebs form when the plasma membrane detaches from the actomyosin cortex. They retract, exerting friction forces and allowing cells to migrate. A few independent studies have reported large and stable blebs in cells under non-adhesive confinement in recent years. This universal switch to bleb-based migration has been found in amoeba, choanoflagellates, immortalized cell lines, and primary cultures. Unlike previously described blebs, they can overcome retraction and <b>stabilize a constant </b>flow. Stable blebs are a new type of cellular structures that amoeboid cells use to migrate, <b>analogous to filopodia or lamellipodia</b> for mesenchymal cells. In a single cell, multiple blebs form and compete against each other, so that eventually, a single bleb drives the migration. Thus, it is essential to know how single blebs stabilize to understand how single-bleb amoeboid cells polarize. More generally, stable actomyosin flows constitute the basis of fast migration in numerous cell types, including also immune cells. During my Ph.D. I studied bleb morphogenesis and bleb stabilization <b>in confined cancer cells</b>, using advanced <b>microfluidic techniques</b> to control the confinement of cells. The first part of my project describes the <b>bleb formation </b>as an immediate response of cells to confinement and what differentiates it from a classical retracting bleb. The second part of my project focuses on the <b>mechanism leading to the establishment of retrograde flow</b>. Based on the results I obtained with my experiments, we propose that bleb stabilization depends on 1) the depletion of actin from the front by myosin contractility and 2) the particular actin filament arrangement at the bleb tip caused by the membrane topology of a confined cell. I completed this work with advanced high-resolution imaging, which allowed observation of single actin filaments and tagged cytoskeleton-associated molecules at the bleb tip, under different perturbations. My results show three cortex regimes in blebs: 1) <b>Assembling loose cortex</b>: localized at the front, composed of single filaments poorly attached to the membrane. If this region is lost, the bleb retracts. 2) <b>Crosslinked cortex</b>: actin filaments and fibers bind together to form a network that gradually gets denser and reticulated but does not contract (this region is devoid of Myosin II motors). 3) <b>Contractile cortex</b>: towards the base of the bleb. Myosin-II starts to get enriched, contracting the dense actin network, driving the entire retrograde actin flow up to the tip of the bleb, generating new actin free regions at the front and pressurizing the bleb, leading to membrane protrusion at the very front. This unique set of observations allowed to complete a model for the stabilization of motile blebs, with conclusions that can be generally applied to any flowing actomyosin cortex. My results present a negative role of actin-membrane attachment in protrusion persistence and contribute to the recent findings in the cell migration field, pointing in this direction. They also introduce an additional role for NMIIA contractility, which is essential for maintaining intracellular pressure and depleting actin from the cell front.