Damage control: new functions for the Arp2/3 complex and WHAMM in autophagy and lysosomal homeostasis

ABSTRACT

Macroautophagy is an intracellular degradation process that relies on autophagosomes and lysosomes to maintain cellular and organismal homeostasis. Actin cytoskeletal rearrangements driven by the Arp2/3 (actin-related protein 2/3) complex, an essential actin nucleator, impact multiple steps of this pathway, but where and when Arp2/3-mediated actin assembly is most influential has remained unclear. Recent work now shows that the Arp2/3 complex is crucial in the later stages of autophagy due to its function in maintaining lysosomal integrity. WHAMM (WASP homolog associated with actin, membranes, and microtubules) is the key nucleation-promoting factor that activates Arp2/3 at permeabilized lysosomes, uncovering new roles for actin, the Arp2/3 complex, and WHAMM in lysosomal damage responses.

Macroautophagy (hereafter autophagy) is a fundamental catabolic process in which intracellular materials are sequestered by autophagosomes that fuse with late endosomes and lysosomes to promote turnover and recycling of the basic components of the structures targeted for degradation. In several autophagic pathways, stressors such as nutrient deprivation lead to inactivation of the metabolic regulator mTORC1 (mechanistic target of rapamycin complex 1) and activation of the ULK1 (unc-51-like autophagy activating kinase 1) and VPS34 (vacuolar protein sorting 34) kinase complexes. This results in the accumulation of the phospholipid phosphatidylinositol-3-phosphate (PtdIns3P) at endoplasmic reticulum-associated omegasomes and nascent isolation membranes termed phagophores. Autophagosome biogenesis is mediated by the lipidation and double-membrane incorporation of ATG8 (autophagy-related 8) protein family members, including LC3A, LC3B, and LC3C (microtubule-associated protein 1 light chain 3 A, B, C), that engage selective autophagy receptors such as p62/SQSTM1 (sequestosome 1) which bind to ubiquitinated targets. The actions of ATG8-family members, tethering factors, and SNARE (soluble NSF attachment protein receptors) molecules enable autophagosomes to fuse with late endosomes and/or lysosomes, where cargo is degraded. Autolysosome tubulation and lysosome reformation can replenish the lysosomal pool in conjunction with TFEB (transcription factor EB), which upregulates lysosome biogenesis genes.

Efficient progression through autophagy requires cytoskeletal rearrangements driven by the Arp2/3 (actin-related protein 2/3) complex and WASP (Wiskott-Aldrich syndrome protein) family of actin nucleation factors (Figure 1). In healthy cells, actin assembled on endolysosomes by the Arp2/3 complex and WASP-family member WASH (WASP and SCAR homolog) promotes anabolic mTORC1 signaling and inhibition of autophagy. However, following starvation or other stimuli, the Arp2/3 complex and WASP-family members WHAMM (WASP homolog associated with actin, membranes, and microtubules) and JMY (junction mediating regulatory) facilitate autophagosome biogenesis through interactions with PtdIns3P and LC3B. WHAMM remains important for the association of p62/SQSTM1 with LC3B during autophagosome closure, and both WHAMM and JMY trigger actin-based motility of autophagosomes. In some forms of autophagy, the Arp2/3 activator Cortactin promotes autophagosome-lysosome fusion. Later, to regenerate lysosomes, WHAMM and Arp2/3 assemble actin filaments that support autolysosome tubulation and autophagic lysosome reformation, while WASH-mediated trafficking activities help maintain a healthy endolysosomal system.

Figure 1.

Schematic depicting the functions of the Arp2/3 complex and WASP-family members in autophagy and lysosomal homeostasis. At steady state, WHAMM is found at endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membranes and JMY is in the nucleus, while WASH and a subset of Arp2/3 complexes localize to lysosomal membranes and promote mTORC1 activation. mTORC1 inhibits nuclear translocation of TFEB and suppresses autophagy by phosphorylating ULK1 (unc-51-like autophagy activating kinase 1), VPS34 (vacuolar protein sorting 34), and other components of the autophagy machinery. During autophagy, WHAMM (WHM) and JMY cooperate with Arp2/3 in autophagosome biogenesis and maturation, Cortactin (CTTN) enhances actin branching during autophagosome-lysosome fusion, and WHAMM supports autolysosome tubulation. Actin filaments are drawn in red and microtubules in green. Our recent study shows that cells lacking the Arp2/3 complex (iKO) harbor chronically damaged lysosomes and fail to complete autophagy. Upon acute lysosomal injury, WHAMM and the Arp2/3 complex are recruited to lysosomes and mitigate damage (box), though their integration with other repair responses remains to be determined.

Although the participation of WASP-family members during different autophagic processes had been well established, we recently sought to identify where and when Arp2/3 complex-driven actin assembly is most important by studying autophagy in mouse fibroblasts with an inducible knockout (iKO) of the essential ArpC2 (Arp complex 2) subunit. This led to the discovery that the Arp2/3 complex and WHAMM enable the completion of autophagy by preserving lysosomal integrity [1].

The first clue that autophagy was dysfunctional without the Arp2/3 complex was that ArpC2 iKO cells accumulated lipidated ATG8 proteins and selective autophagy receptors including p62/SQSTM1 and Optineurin under normal growth conditions. A tandem-fluorescent LC3 reporter further revealed that iKO cells failed to undergo proper autolysosome acidification and turnover. A key observation was that the permeabilized lysosome sensor Galectin-3 and the ESCRT-III (endosomal sorting complex required for transport III) membrane-repair component IST1 (increased sodium tolerance 1) were present at lysosomes in iKO cells, suggesting that those lysosomes were damaged. Pharmacological Arp2/3 inhibition and washout studies then demonstrated that lysosomes were hypersensitive to the endolysosomal-damaging agent LLOMe (L-leucyl-L-leucine methyl ester) and unable to repair their membranes without a functional Arp2/3 complex.

We next asked which proteins collaborate with Arp2/3 to ensure lysosomal integrity by studying a panel of human fibroblasts lacking each known actin nucleation-promoting factor. These experiments implicated WHAMM, JMY, and WASH as key players in the lysosomal damage response. Immunofluorescence microscopy revealed that JMY remained primarily nuclear in the presence of LLOMe, whereas WASH localization to lysosomes decreased. Most notably, WHAMM was recruited to nearly every lysosome following LLOMe treatment. We concluded that WHAMM is the Arp2/3 activator with the most direct function in the lysosomal damage response.

Lastly, we visualized the Arp2/3 complex in WHAMM-expressing versus WHAMM-knockout mouse fibroblasts. Without WHAMM, the lysosomal localization of ArpC2 and Arp3 was reduced, establishing that WHAMM is crucial for Arp2/3 complex recruitment to lysosomes upon their damage. Interestingly, male WHAMM-knockout cells were more sensitive than female WHAMM-knockout cells to LLOMe, suggesting that cell-intrinsic sex differences impact lysosomal homeostasis. Collectively, our results highlight the Arp2/3 complex and WHAMM as major players in maintaining lysosome integrity.

These findings add a new layer of complexity to the cytoskeletal functions that enable autophagy, because without adequate lysosomes to complete the degradation process, the positive effects of actin assembly earlier in autophagosome biogenesis and maturation would be futile. The data also raise many questions about how cytoskeletal activities are regulated and coordinated with other membrane trafficking pathways. For example, do the lysosomal defects in Arp2/3-deficient cells also prevent endocytic degradation? WASH is a key figure in endosome remodeling, but how and why is WASH removed from damaged lysosomes? Moreover, does JMY ever localize to damaged lysosomes, or does it only exert its pro-lysosome activity from within the nucleus? JMY regulates transcription during other stress responses, so it is intriguing to speculate that JMY might cooperate with TFEB or other factors to upregulate a transcriptional program for lysosome biogenesis.

Additional questions are centered around WHAMM, the Arp2/3 complex, and actin itself. What are the molecular mechanisms underlying WHAMM’s recognition of damaged lysosomes? WHAMM interacts with microtubules, phosphoinositides, and G-proteins. But whether these or other binding-partners influence its lysosomal localization remains to be explored. Further, why is WHAMM more important in male cells, and are sex-specific lysosomal phenotypes related to the kidney dysfunction found in male WHAMM-knockout mice?

Recent investigations into lysosomal repair have revealed diverse responses including membrane remodeling by ESCRT complexes, lipid transfer at endoplasmic reticulum contact sites, ATG8 conjugation, autophagic lysosomal reformation pathways, sphingomyelin and annexin reorganization, and stress granule condensation. Future studies are needed to define where WHAMM and Arp2/3 intersect with those mechanisms or with different repair pathways. Moreover, when damage is irreparable, lysosomes can be removed via lysophagy to avoid cell death. Perhaps, the actin nucleation factors coordinate the transition from lysosome repair to lysophagy or apoptosis by revisiting their roles in autophagosome biogenesis or shifting to their functions in apoptotic signaling. But is the only essential activity of WHAMM to promote Arp2/3-mediated actin assembly, or does WHAMM have Arp2/3-independent activities? Finally, do actin filaments physically stabilize the membrane of the lysosome, help contain its contents, act as a scaffold for other proteins, or perform some other function? A deeper mechanistic understanding of how cytoskeletal factors engage lysosomes and integrate with different repair machineries will provide a more holistic view on how lysosomal homeostasis is preserved.

Funding Statement

The work was supported by the National Institutes of Health [GM155651].

Abbreviations

Arp2/3: actin-related protein 2/3; ArpC2: Arp complex subunit 2; ATG8: autophagy-related 8; ERGIC: endoplasmic reticulum-Golgi intermediate compartment; ESCRT-III: endosomal sorting complex required for transport III; iKO: inducible knockout; IST1: increased sodium tolerance 1; JMY: junction mediating and regulatory; LC3: microtubule-associated protein 1 light chain 3; LLOMe: L-leucyl-L-leucine methyl ester; mTORC1: mechanistic target of rapamycin complex 1; PtdIns3P: phospholipid phosphatidylinositol-3-phosphate; p62/SQSTM1: sequestosome 1; SNARE: soluble NSF attachment protein receptor; TFEB: transcription factor EB; ULK1: unc-51-like autophagy activating kinase 1; VPS34: vacuolar protein sorting 34; WASH: WASP and SCAR Homolog; WASP: Wiskott-Aldrich Syndrome Protein; WHAMM: WASP Homolog associated with Actin, Membranes, and Microtubules.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data sharing

No data sharing is applicable in this case as no new data has been analyzed or created in this study.