Structural insights into WHAMM-mediated cytoskeletal coordination during membrane remodeling

Summary Nucleation promoting factors (NPFs) control the spatio-temporal activity of Arp2/3 complex in cells [1, 2]. Thus, WASP and the WAVE complex direct the formation of branched actin networks at the leading edge during cell motility and endo/exocytosis, whereas the WASH complex is involved in endosomal transport. Less understood are WHAMM and JMY, two NPFs with similar domain architecture. JMY is found in the nucleus and the cytosol, and is involved in transcriptional regulation [3], cell motility [4], and trans-Golgi transport [5]. WHAMM was reported to bind microtubules and to be involved in ER to cis-Golgi transport [6]. Here, we show that WHAMM directs the activity of Arp2/3 complex for autophagosome biogenesis through an actin-comet tail motility mechanism. Macroautophagy – the process by which cytosolic material is engulfed into autophagosomes for degradation and/or recycling – was recently shown to involve actin [7], but the mechanism is unknown. We found that WHAMM forms puncta that colocalize and comigrate with the autophagy markers LC3, DFCP1 and p62 through a WHAMM-dependent actin-comet tail mechanism. Under starvation, WHAMM and actin are observed at the interface between neighboring autophagosomes, whose number and size increases with WHAMM expression. Interfering with actin polymerization, inhibiting Arp2/3 complex, knocking down WHAMM, or blocking its interaction with Arp2/3 complex through mutagenesis, all inhibit comet tail formation and reduce the size and number of autophagosomes. Finally, JMY shows similar localization to WHAMM, and could be involved in similar processes. These results reveal a link between Arp2/3 complex-dependent actin assembly and autophagy.

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“…Thus, the middle region of WHAMM (aa 174–541), implicated in MT binding [17], seems to be at least in part responsible for this phenotype.…”

Section: Resultsmentioning

confidence: 99%

WHAMM Directs the Arp2/3 Complex to the ER for Autophagosome Biogenesis through an Actin Comet Tail Mechanism

et al. 2015

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“…Similar images are also obtained with other MT-binding proteins, although their biochemical analyses and established biological functions do not involve a high degree of cooperativity. 34,37 It seems likely that a high heterogeneity of MT decoration reported by cryo-electron microscopy might be an artifact of some aspect of a sample preparation procedure. Our experimental work with GFP-labeled NDC80 proteins shows the homogeneous decoration of MTs when experiments are carried out with reagents that block the non-specific protein binding to a surface of microscopy chamber, but highly heterogeneous MT decoration is seen when such blocking reagents are omitted (unpublished observation).…”

Section: Resultsmentioning

confidence: 99%

Highly Transient Molecular Interactions Underlie the Stability of Kinetochore–Microtubule Attachment During Cell Division

2013

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Chromosome segregation during mitosis is mediated by spindle microtubules that attach to chromosomal kinetochores with strong yet labile links. The exact molecular composition of the kinetochore–microtubule interface is not known but microtubules are thought to bind to kinetochores via the specialized microtubule-binding sites, which contain multiple microtubule-binding proteins. During prometaphase the lifetime of microtubule attachments is short but in metaphase it increases 3-fold, presumably owing to dephosphorylation of the microtubule-binding proteins that increases their affinity. Here, we use mathematical modeling to examine in quantitative and systematic manner the general relationships between the molecular properties of microtubule-binding proteins and the resulting stability of microtubule attachment to the protein-containing kinetochore site. We show that when the protein connections are stochastic, the physiological rate of microtubule turnover is achieved only if these molecular interactions are very transient, each lasting fraction of a second. This “microscopic” time is almost four orders of magnitude shorter than the characteristic time of kinetochore–microtubule attachment. Cooperativity of the microtubule-binding events further increases the disparity of these time scales. Furthermore, for all values of kinetic parameters the microtubule stability is very sensitive to the minor changes in the molecular constants. Such sensitivity of the lifetime of microtubule attachment to the kinetics and cooperativity of molecular interactions at the microtubule-binding site may hinder the accurate regulation of kinetochore–microtubule stability during mitotic progression, and it necessitates detailed experimental examination of the microtubule-binding properties of kinetochore-localized proteins.

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“…More recent studies have shown that RhoD interacts with WHAMM to regulate Golgi structure and the transport of cargo to the plasma membrane (Blom et al, 2015;Gad et al, 2012). Cryoelectron microscopy revealed that WHAMM binds to the outer surface of MT protofilaments via its CC domain and forms helical head-to-tail structures along MTs (Shen et al, 2012). MT binding induces a structural change that exposes the N-terminal WMD for membrane binding and tubulation activity, while masking the C-terminal WWCA motif, thus preventing actin polymerization (Liu et al, 2017).…”

Section: Regulation and Cytoplasmic Functions Of Whamm Jmy And Wash mentioning

confidence: 99%

Cellular functions of WASP family proteins at a glance

Alekhina

Burstein

Billadeau

2017

Journal of Cell Science

165

172

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Proteins of the Wiskott–Aldrich syndrome protein (WASP) family function as nucleation-promoting factors for the ubiquitously expressed Arp2/3 complex, which drives the generation of branched actin filaments. Arp2/3-generated actin regulates diverse cellular processes, including the formation of lamellipodia and filopodia, endocytosis and/or phagocytosis at the plasma membrane, and the generation of cargo-laden vesicles from organelles including the Golgi, endoplasmic reticulum (ER) and the endo-lysosomal network. Recent studies have also identified roles for WASP family members in promoting actin dynamics at the centrosome, influencing nuclear shape and membrane remodeling events leading to the generation of autophagosomes. Interestingly, several WASP family members have also been observed in the nucleus where they directly influence gene expression by serving as molecular platforms for the assembly of epigenetic and transcriptional machinery. In this Cell Science at a Glance article and accompanying poster, we provide an update on the subcellular roles of WHAMM, JMY and WASH (also known as WASHC1), as well as their mechanisms of regulation and emerging functions within the cell.

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Structural insights into WHAMM-mediated cytoskeletal coordination during membrane remodeling

Abstract: Structural analysis of the WHAMM–microtubule interaction provides insight into WHAMM’s coordination of microtubule binding, membrane association, and actin nucleation.

Cited by 31 publications

References 62 publications

WHAMM Directs the Arp2/3 Complex to the ER for Autophagosome Biogenesis through an Actin Comet Tail Mechanism

WHAMM Directs the Arp2/3 Complex to the ER for Autophagosome Biogenesis through an Actin Comet Tail Mechanism

Highly Transient Molecular Interactions Underlie the Stability of Kinetochore–Microtubule Attachment During Cell Division

Cellular functions of WASP family proteins at a glance

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