Regulation of JMY’s actin nucleation activity by TTC5/STRAP and LC3 during autophagy

ABSTRACT

Actin plays indispensable roles in autophagosome biogenesis. Branched actin networks assembled within phagophore membranes are required for generating the autophagosome membrane shape and movement. The ARP2/3 complex and its regulators, such as JMY (junction mediating and regulatory protein, p53 cofactor), translocate to phagophore membranes to promote local actin filament formation during autophagy. Hu et al., recently showed that during autophagy LC3 recruits JMY to the phagophore and promotes its actin nucleation activity. They also characterized TTC5/STRAP (tetratricopeptide repeat domain 5) as a negative autophagy regulator via binding to JMY and antagonizing its activation. Moreover, an in vitro reconstitution system was developed to demonstrate that membrane-bound LC3 is sufficient to recruit JMY and stimulate JMY-mediated actin filament assembly.

JMY was first identified as a cofactor for EPS300/p300, regulating the TP53/p53 response during DNA damage [1]. Although JMY can function in the nucleus, it localizes mostly to the cytoplasm in cells under normal conditions; cytosolic JMY regulates actin filament formation. The C terminus of JMY contains a WCA domain present in class II actin nucleation-promoting factors. This domain mediates its binding to both the ARP2/3 complex and actin, promoting ARP2/3-depdent actin nucleation [2]. Recently, JMY was reported to promote autophagy by facilitating actin assembly on autophagic membranes [3]. The N terminus of JMY contains an LC3-interacting region (LIR) that is required for its translocation to the phagophore and its positive role in autophagy. However, little is known about how JMY’s actin nucleation activity and its role in autophagy are regulated. In the recent study by Hu et al, the authors reported that both TTC5/STRAP and LC3 regulate JMY-mediated actin filament assembly on phagophore and autophagosome membranes [4].

In the nucleus, TTC5/STRAP interacts with JMY to prevent its degradation [5]; however, a pool of TTC5/STRAP also localizes in the cytoplasm. Accordingly, the authors decided to test whether TTC5/STRAP is involved in regulating JMY’s function during autophagy. In fed U2OS cells, JMY and TTC5/STRAP are present mostly in cytoplasmic foci, and the 2 proteins significantly colocalize with each other. Consistent with the previous study [3], JMY translocates to LC3-containing membranes after autophagy induction by starvation. In contrast, the colocalization between JMY and TTC5/STRAP significantly decreases under this condition. These observations suggest that upon starvation, LC3 competes with TTC5/STRAP for binding to JMY and recruits JMY to autophagic membranes. Moreover, by tracing the LC3-labeled membranes over time, the authors found that the ones with both JMY and LC3, but without TTC5/STRAP, are very motile, whereas those containing JMY and TTC5/STRAP display relatively little movement, indicating that TTC5/STRAP inhibits the motility of JMY-localized membranes.

JMY promotes actin assembly on LC3-positive autophagic membranes [3]. One obvious question is whether actin filaments are required for the motility of these membranes. Using 3-color microscopy, the authors found that the LC3- and JMY-positive vesicles are associated with filamentous actin. When the wild-type cells are treated with CK666, a drug that inhibits nucleation activity of the ARP2/3 complex, the LC3- and JMY-containing membranes become nonmotile. In addition, in the JMY knockout cells, the majority of LC3 puncta are largely not motile. These data collectively imply that the motility of LC3-containing membranes requires JMY- and ARP2/3-mediated actin filament assembly. Moreover, in cells overexpressing TTC5/STRAP, LC3-positive vesicles show significantly decreased motility, suggesting that TTC5/STRAP negatively regulates JMY and ARP2/3 function. This observation led to the hypothesis that TTC5/STRAP is a negative regulator of autophagy. In support of this idea, in TTC5/STRAP knockout cells, the authors observed significantly increased colocalization between LC3 and JMY, increased number and size of autophagosomes, and decreased SQSTM1/p62 levels, compared to that in the wild-type cells under starvation conditions.

To test whether TTC5/STRAP inhibits autophagy by directly antagonizing the actin nucleation activity of JMY, the authors purified TTC5/STRAP, JMY and the ARP2/3 complex, and examined branched actin formation when different combinations of proteins were added into the system. They found that TTC5/STRAP not only inhibits the intrinsic actin nucleation activity of JMY, but also diminishes its ability to promote ARP2/3-mediated actin nucleation. Using the same method, the authors revealed that in contrast to TTC5/STRAP, LC3 enhances JMY’s actin nucleation activity with or without the presence of the ARP2/3 complex. Moreover, liposome-bound LC3 even more robustly stimulates JMY’s intrinsic and ARP2/3-dependent actin nucleation activities than soluble LC3.

Finally, the authors reconstituted LC3- and JMY-dependent actin filament formation from purified components in vitro. With this system, they showed that glass microspheres coated with LC3 are able to recruit JMY, and that in the presence of necessary factors such as the ARP2/3 complex, initiation of branched actin assembly is induced. In agreement with the in vivo data, when TTC5/STRAP is present in the system, it suppresses the formation of actin networks. Overall, the results presented in the study support a model in which autophagic membrane-localized LC3 recruits and stimulates JMY’s ability to promote local actin assembly, which is necessary for the motility of these membranes, while TTC5/STRAP inhibits this process by competing with LC3 for binding to JMY and antagonizing its actin nucleation activity.

This study sheds more light on how local actin assembly mediated by JMY and the ARP2/3 complex regulates auto-phagic membrane motility. It also identifies TTC5/STRAP as a novel autophagy regulator through modulating JMY’s actin nucleation activity. Moreover, reconstitution of the LC3- and JMY-mediated actin network formation in vitro provides the first demonstration that membrane-bound LC3 is sufficient to recruit JMY and activate JMY-dependent actin assembly.

Funding Statement

This work was supported by the National Institute of General Medical Sciences [GM053396].

Disclosure statement

No potential conflict of interest was reported by the authors.