ROCK1 but not LIMK1 or PAK2 is a key regulator of apoptotic membrane blebbing and cell disassembly

Abstract

Many cell types are known to undergo a series of morphological changes during the progression of apoptosis, leading to their disassembly into smaller membrane-bound vesicles known as apoptotic bodies (ApoBDs). In particular, the formation of circular bulges called membrane blebs on the surface of apoptotic cells is a key morphological step required for a number of cell types to generate ApoBDs. Although apoptotic membrane blebbing is thought to be regulated by kinases including ROCK1, PAK2 and LIMK1, it is unclear whether these kinases exhibit overlapping roles in the disassembly of apoptotic cells. Utilising both pharmacological and CRISPR/Cas9 gene editing based approaches, we identified ROCK1 but not PAK2 or LIMK1 as a key non-redundant positive regulator of apoptotic membrane blebbing as well as ApoBD formation. Functionally, we have established an experimental system to either inhibit or enhance ApoBD formation and demonstrated the importance of apoptotic cell disassembly in the efficient uptake of apoptotic materials by various phagocytes. Unexpectedly, we also noted that ROCK1 could play a role in regulating the onset of secondary necrosis. Together, these data shed light on both the mechanism and function of cell disassembly during apoptosis.

Unleashing the therapeutic potential of apoptotic bodies

“…Of many mechanisms that aid the cell clearance process [17,18], the disassembly of apoptotic cells into 'bite-sized' ApoBDs could mediate the rapid and efficient debris removal via efferocytosis, by tissue-resident professional phagocytes (e.g. macrophages and immature dendritic cells) or by neighbouring non-professional phagocytes [12,13,19]. Impaired ApoBD release and consequential defective apoptotic cell clearance have been associated with inflammation and autoimmunity due to subsequent cell/ApoBD lysis as well as exposure of autoantigens ( Figure 2) [13,17,20,21].…”

“…Nevertheless, recent efforts in establishing ApoBD characterisation criteria and developing ApoBD research tools have led to further insights into ApoBDs biogenesis and function [10,11]. It is becoming apparent that ApoBDs are key messengers released by dying cells to regulate processes including cell clearance, tissue homeostasis, pathogen dissemination and immunity, thus implicating its therapeutic potential [1,[12][13][14][15]. In this review, we discuss this exciting, yet underappreciated, aspect of ApoBDs, thereby positioning ApoBDs as another candidate in EV's race to therapeutic application.…”

Stoking the Fire: How Dying Cells Propagate Inflammatory Signalling through Extracellular Vesicle Trafficking

“…ApoBDs are typically 1–5 μm in size and are generated solely during apoptosis in a caspase-3/7-mediated step-wise fragmentation process known as “apoptotic cell disassembly”. Initial plasma membrane blebbing, regulated by rho-associated coiled-coil associated kinase 1 (ROCK1), is followed by the formation of plasma membrane protrusions, regulated positively by plexin B2 (PlexB2) and negatively by pannexin 1 (PANX1) [ 59 , 60 , 61 ]. Finally, the release of distinct ApoBDs from the cell occurs [ 60 ].…”

Apoptotic cells secrete metabolites to regulate immune homeostasis

“…These observations suggest that additional mechanisms must exist to aid the release of the remaining 80% of metabolites from apoptotic cells, possibly via other membrane channels with activity regulated (directly or indirectly) by caspase activation (Figure 1). Caspase‐activated Panx1 has been described to regulate a broad range of processes during apoptosis, including the recruitment of phagocytes toward dying cells though the release of “find‐me” signals adenosine triphosphate and uridine triphosphate, 7 the immune response through the release of “calm‐down” signal adenosine monophosphate 10 and other metabolites (as will be detailed later), the disassembly of apoptotic cells into fragments known as apoptotic bodies to aid cell clearance 11,12 and NLRP3 inflammasome assembly 13 . Precisely how these Panx1‐regulated processes intertwine to modulate cell clearance and immunity remains to be fully defined.…”