An Amish founder mutation disrupts a PI(3)P-WHAMM-Arp2/3 complex–driven autophagosomal remodeling pathway

Mathiowetz, Alyssa J.; Baple, Emma L.; Russo, Ashley J.; Coulter, Alyssa; Carrano, Eric; Brown, Judith; Jinks, Robert N.; Crosby, Andrew H.; Campellone, Kenneth

doi:10.1091/mbc.e17-01-0022

Cited by 26 publications

(56 citation statements)

References 51 publications

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“…This lower potency likely stems from a fewer (than in formins and Ena/VASP) number of G-actin binding domains and, possibly, a less favorable geometry for oligomer binding to NPFs. Of note, depending on the involvement in a particular cellular process (i.e., remodeling of cytoplasmic [31], endosomal [32], autophagosomal [33, 34], Golgi and ER membranes [35, 36], or contribution to cytoplasmic streaming [37], etc. ), the Arp2/3 complex can be activated by different NPFs, of which only two were tested in the present study: VCA fragment of N-WASP in the in vitro assays (Figures 2G–O) and WAVE in the cellular context (Figures 2D–F).…”

Section: Discussionmentioning

confidence: 99%

Actin Cross-Linking Toxin Is a Universal Inhibitor of Tandem-Organized and Oligomeric G-Actin Binding Proteins

et al. 2018

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Delivery of bacterial toxins to host cells is hindered by host protective barriers. This obstruction dictates a remarkable efficiency of toxins, a single copy of which may kill a host cell. Efficiency of actin-targeting toxins is further hampered by an overwhelming abundance of their target. The actin cross-linking domain (ACD) toxins of Vibrio species and related bacterial genera catalyze the formation of covalently cross-linked actin oligomers. Recently, we reported that the ACD toxicity can be amplified via a multivalent inhibitory association of actin oligomers with actin assembly factors formins, suggesting that the oligomers may act as secondary toxins. Importantly, many proteins involved in nucleation, elongation, severing, branching, and bundling of actin filaments contain G-actin-binding Wiskott-Aldrich syndrome protein (WASP)-homology motifs 2 (WH2) organized in tandem and therefore may act as a multivalent platform for high-affinity interaction with the ACD-cross-linked actin oligomers. Using live-cell single-molecule speckle (SiMS) microscopy, total internal reflection fluorescence (TIRF) microscopy, and actin polymerization assays, we show that, in addition to formins, the oligomers bind with high affinity and potently inhibit several families of actin assembly factors: Ena/vasodilator-stimulated phosphorprotein (VASP); Spire; and the Arp2/3 complex, both in vitro and in live cells. As a result, ACD blocks the actin retrograde flow and membrane dynamics and disrupts association of Ena/VASP with adhesion complexes. This study defines ACD as a universal inhibitor of tandem-organized G-actin binding proteins that overcomes the abundance of actin by redirecting the toxicity cascade toward less abundant targets and thus leading to profound disorganization of the actin cytoskeleton and disruption of actin-dependent cellular functions.

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Section: Discussionmentioning

confidence: 99%

Actin Cross-Linking Toxin Is a Universal Inhibitor of Tandem-Organized and Oligomeric G-Actin Binding Proteins

et al. 2018

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“…Nucleation factors from the WASP family assimilate upstream signals and activate the Arp2/3 complex to drive actin assembly. The physiological importance of these factors is underscored by the observations that murine N-WASP, WAVE1, WAVE2, and WASH gene knockouts result in lethality [18, 19, 21, 28], that mutations in human Wasp give rise to immunodeficiencies [17, 83], and that a mutation in Whamm is found in patients with a neurodevelopmental and renal disorder [37, 84]. All family members have been shown to influence cell motility, with at least 6 playing roles in membrane protrusion.…”

Section: Discussionmentioning

confidence: 99%

“…WASH [27] is essential in mice [28], likely due to its role in directing endo-lysosome trafficking [29-31]. WHAMM and JMY both drive the remodeling or transport of membranes in the secretory pathway [32-34] as well as autophagosomes [35-37]. WASH and WHAMM can indirectly affect cell motility [38-40], likely due to their functions in membrane trafficking, while JMY can be recruited to the front of motile cells and accelerate wound healing migration [41].…”

Section: Introductionmentioning

confidence: 99%

WHIMP links the actin nucleation machinery to Src-family kinase signaling during protrusion and motility

Kabrawala

Zimmer

Campellone

2020

Preprint

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17Cell motility is governed by cooperation between the Arp2/3 complex and nucleation factors 18 from the Wiskott-Aldrich Syndrome Protein (WASP) family, which together assemble actin 19 filament networks to drive membrane protrusion. Here we identify WHIMP (WAVE Homology In 20Membrane Protrusions) as a new member of the WASP family. The Whimp gene is encoded on 21 the X-chromosome of multiple animals, including mice. Murine WHIMP promotes Arp2/3-22 dependent actin assembly, but is less potent than other nucleation factors. Nevertheless, 23 WHIMP-mediated Arp2/3 activation enhances both plasma membrane ruffling and wound 24 healing migration, whereas WHIMP depletion impairs protrusion and slows motility. WHIMP 25 expression also increases Src-family kinase activity, and WHIMP-induced ruffles contain the 26 additional nucleation factors WAVE1, WAVE2, and N-WASP, but not JMY or WASH. Perturbing 27 the function of Src-family kinases, WAVE proteins, or Arp2/3 complex inhibits WHIMP-driven 28 ruffling. These results suggest that WHIMP-mediated actin assembly plays a direct role in 29 membrane protrusion, but also results in feedback control of tyrosine kinase signaling to 30 modulate the activation of multiple WASP-family proteins. 31 32 33 3 AUTHOR SUMMARY 34The actin cytoskeleton is a collection of protein polymers that assemble and disassemble within 35 cells at specific times and locations. Sophisticated cytoskeletal regulators called nucleation 36 factors ensure that actin polymerizes when and where it is needed, and most nucleation factors 37 are members of the Wiskott-Aldrich Syndrome Protein (WASP) family. Several of the 8 known 38WASP-family proteins function in cell motility, but how the different factors collaborate with one 39 another is not well understood. In this study, we identified WHIMP, a new WASP-family 40 member which is encoded on the X chromosome of a variety of animals. In mouse cells, 41 WHIMP enhances cell motility by assembling actin filaments that push the cell membrane 42 forward. Unexpectedly, WHIMP also activates tyrosine kinase enzymes, proteins that stimulate 43 multiple WASP-family members during motility. Our results open new avenues of research into 44 how nucleation factors cooperate during movement and how the molecular activities that 45 underlie motility differ in distinct cell types and organisms. 46 47 48 the recruitment and activation of a 7-subunit macromolecule named the Arp2/3 complex [2], 52 which acts in concert with binding partners called nucleation-promoting factors [3]. Many such 53 factors are members of the Wiskott-Aldrich Syndrome Protein (WASP) family, and are integral in 54 activating the complex at different cellular locations [4]. Most WASP-family proteins promote 55 actin assembly during membrane protrusion and cell motility [5], but how the different factors 56 collaborate during these processes is not well understood. 57 WASP-family members are defined by the presence of a WH2-Connector-Acidic (WCA) 58 domain in which one or more WH2 motifs bind actin monome...

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“…Once detached, autophagosomes must move to and fuse with lysosomes/late endosomes. WHAMM is recruited to membranes by an early event in phagophore initiation: phosphorylation of phosphatidyl inositol in the endoplasmic reticulum to form PI(3)P. This acidic phospholipid binds and recruits WHAMM via interaction with PX domain-like sequences in the N-terminal region of the protein (Mathiowetz et al, 2017). In contrast, JMY interacts with LC3, whose lipidation happens after WHAMM is recruited to PI3P sites (Mathiowetz et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

“…WHAMM is recruited to membranes by an early event in phagophore initiation: phosphorylation of phosphatidyl inositol in the endoplasmic reticulum to form PI(3)P. This acidic phospholipid binds and recruits WHAMM via interaction with PX domain-like sequences in the N-terminal region of the protein (Mathiowetz et al, 2017). In contrast, JMY interacts with LC3, whose lipidation happens after WHAMM is recruited to PI3P sites (Mathiowetz et al, 2017). This might suggest that JMY is strictly downstream of WHAMM, but the story is not so simple because we also observe colocalization and co-migration of JMY with proteins involved in the initiation and nucleation phases of phagophore assembly (DFCP1, ATG9) ( Figure S5B-C).…”

Section: Discussionmentioning

confidence: 99%

LC3 and STRAP regulate actin filament assembly by JMY during autophagosome formation

Hu¹,

Mullins²

2018

Preprint

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The actin regulator JMY creates filament networks that move membranes during autophagy. We find that, in unstarved cells, JMY is inhibited by interaction with the STRAP protein, but upon starvation JMY is recruited away from STRAP and activated by LC3. AbstractDuring autophagy actin filament networks move and remodel cellular membranes to form autophagosomes that enclose and metabolize cytoplasmic contents. Two actin regulators, WHAMM and JMY, participate in autophagosome formation, but the signals linking autophagy to actin assembly are poorly understood. We show that, in non-starved cells, cytoplasmic JMY co-localizes with STRAP, a regulator of JMY's nuclear functions, on non-motile vesicles with no associated actin networks. Upon starvation, JMY shifts to motile, LC3-containing membranes that move on actin comet tails. LC3 enhances JMY's de novo actin nucleation activity via a cryptic actin-binding sequence near JMY's Nterminus, and STRAP inhibits JMY's ability to nucleate actin and activate the Arp2/3 complex. Cytoplasmic STRAP negatively regulates autophagy. Finally, we use purified proteins to reconstitute LC3-and JMY-dependent actin network formation on membranes, and inhibition of network formation by STRAP. We conclude that LC3 and STRAP regulate JMY's actin assembly activities in trans during autophagy.

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An Amish founder mutation disrupts a PI(3)P-WHAMM-Arp2/3 complex–driven autophagosomal remodeling pathway

Cited by 26 publications

References 51 publications

Actin Cross-Linking Toxin Is a Universal Inhibitor of Tandem-Organized and Oligomeric G-Actin Binding Proteins

Actin Cross-Linking Toxin Is a Universal Inhibitor of Tandem-Organized and Oligomeric G-Actin Binding Proteins

WHIMP links the actin nucleation machinery to Src-family kinase signaling during protrusion and motility

LC3 and STRAP regulate actin filament assembly by JMY during autophagosome formation

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