The actin nucleation factors JMY and WHAMM enable a rapid Arp2/3 complex-mediated intrinsic pathway of apoptosis

Abstract

The actin cytoskeleton is a well-known player in most vital cellular processes, but comparably little is understood about how the actin assembly machinery impacts programmed cell death pathways. In the current study, we explored roles for the human Wiskott-Aldrich Syndrome Protein (WASP) family of actin nucleation factors in DNA damage-induced apoptosis. Inactivation of each WASP-family gene revealed that two of them, JMY and WHAMM, are necessary for rapid apoptotic responses. JMY and WHAMM participate in a p53-dependent cell death pathway by enhancing mitochondrial permeabilization, initiator caspase cleavage, and executioner caspase activation. JMY-mediated apoptosis requires actin nucleation via the Arp2/3 complex, and actin filaments are assembled in cytoplasmic territories containing clusters of cytochrome c and active caspase-3. The loss of JMY additionally results in significant changes in gene expression, including upregulation of the WHAMM-interacting G-protein RhoD. Depletion or deletion of RHOD increases cell death, suggesting that RhoD normally contributes to cell survival. These results give rise to a model in which JMY and WHAMM promote intrinsic cell death responses that can be opposed by RhoD.

F-actin-rich territories coordinate apoptosome assembly and caspase activation during intrinsic apoptosis

“…Apoptotic F-actin-rich territories contain the JMY/WHAMM subfamily of Arp2/3-activating nucleation factors JMY, its closest homolog WHAMM, and their downstream branched actin nucleator, the Arp2/3 complex, are all required for efficient intrinsic apoptosis (King et al, 2021). Therefore, we next studied the localization of these and other actin nucleation and branching factors in relation to the F-actin-rich territories.…”

“…Genetic inactivation of each WASP-family Arp2/3 activator previously revealed that only cells lacking JMY or WHAMM have substantial deficiencies in their intrinsic apoptosis responses (King et al, 2021). To characterize which, if any, WASP-family proteins other than JMY associate with the apoptotic F-actin territories, we treated U2OS cells with etoposide and assessed the localization of WHAMM and representatives of other subgroups within the WASP family.…”

“…Localized JMY-mediated Arp2/3 activation and actin polymerization are crucial for driving executioner caspase activation While previous work showed that JMY requires its actin-and Arp2/3-binding domains in order to enable efficient caspase-3 cleavage (King et al, 2021), the degree to which actin assembly specifically within territories impacts caspase-3 processing is unknown. JMY activates the Arp2/3 complex using a C-terminal domain comprising 3 actin-binding WASP-homology-2 (W) motifs and an Arp2/3-binding Connector Acidic (CA) segment, and can also use its WWW region to nucleate actin directly (Zuchero et al, 2009).…”

“…JMY activates the Arp2/3 complex using a C-terminal domain comprising 3 actin-binding WASP-homology-2 (W) motifs and an Arp2/3-binding Connector Acidic (CA) segment, and can also use its WWW region to nucleate actin directly (Zuchero et al, 2009). JMY knockout (JMY KO ) fibroblasts are deficient in caspase-3 activation (King et al, 2021), so we utilized 3 GFP-tagged JMY derivatives for performing rescue experiments: wild type JMY (JMY WT ); a JMY mutant missing all 3 W motifs (JMY ΔWWW ) that can neither nucleate actin directly nor cooperate with the Arp2/3 complex; and a JMY mutant lacking the CA segment (JMY ΔCA ) that cannot activate Arp2/3 but retains its actin nucleating WWW region. We introduced the GFP-JMY plasmids, or a vector control, into the JMY KO cells, exposed them to etoposide, and stained them to visualize active CCasp-3 and F-actin (Figure 5A).…”

“…JMY shuttles between the nucleus and cytosol (Coutts et al, 2009; Zuchero et al, 2012), and although it may have pro-apoptotic functions in the nucleus, its most extensively characterized activities are as a cytosolic actin nucleation factor (Zuchero et al, 2009). In fact, following DNA damage, JMY, its closest homolog WHAMM, and the Arp2/3 complex are necessary for rapid pro-apoptotic responses in the cytoplasm (King et al, 2021). Cells lacking JMY or WHAMM exhibit kinetic delays or deficiencies in the mitochondrial export of cyto c , activation of caspases, and ultimately the terminal cleavage events throughout the cell (King et al, 2021).…”