Cellular morphogenesis and processes such as cell division and migration require the coordination of the microtubule and actin cytoskeletons. Microtubule–actin crosstalk is poorly understood and largely regarded as the capture and regulation of microtubules by actin. Septins are filamentous guanosine-5'-triphosphate (GTP) binding proteins, which comprise the fourth component of the cytoskeleton along microtubules, actin, and intermediate filaments. Here, we report that septins mediate microtubule–actin crosstalk by coupling actin polymerization to microtubule lattices. Superresolution and platinum replica electron microscopy (PREM) show that septins localize to overlapping microtubules and actin filaments in the growth cones of neurons and non-neuronal cells. We demonstrate that recombinant septin complexes directly crosslink microtubules and actin filaments into hybrid bundles. In vitro reconstitution assays reveal that microtubule-bound septins capture and align stable actin filaments with microtubules. Strikingly, septins enable the capture and polymerization of growing actin filaments on microtubule lattices. In neuronal growth cones, septins are required for the maintenance of the peripheral actin network that fans out from microtubules. These findings show that septins directly mediate microtubule interactions with actin filaments, and reveal a mechanism of microtubule-templated actin growth with broader significance for the self-organization of the cytoskeleton and cellular morphogenesis.
Cellular morphogenesis and processes such as cell division and migration require the coordination of the microtubule and actin cytoskeletons. Microtubule-actin crosstalk is poorly understood and largely regarded as the capture and regulation of microtubules by actin. Septins are filamentous GTP-binding proteins, which comprise the fourth component of the cytoskeleton along microtubules, actin and intermediate filaments. Here, we report that septins mediate microtubule-actin crosstalk by coupling actin polymerization to microtubule lattices. Super-resolution imaging shows that septins localize to overlapping microtubules and actin filaments in the growth cones of neurons and non-neuronal cells. We show that recombinant septin complexes directly crosslink microtubules and actin filaments into hybrid bundles. In vitro reconstitution assays reveal that microtubule-bound septins capture and align stable actin filaments with microtubules. Strikingly, septins enable the capture and polymerization of growing actin filaments on microtubule lattices. In neuronal growth cones, septins are required for the maintenance of the peripheral actin network that fans out from microtubules. These findings provide the first evidence of septins directly mediating microtubule interactions with actin filaments, and reveal a new mechanism of microtubule-templated actin growth with broader significance for the self-organization of the cytoskeleton and cellular morphogenesis.
The binder of rho GTPases (BORG)/Cdc42 effector proteins (Cdc42EP) family is composed of five Rho GTPase binding proteins whose functions and mechanism of actions are of emerging interest. Here, we review recent findings pertaining to the family as a whole and consider how these change our understanding of cellular organization. Recent studies have implicated BORGs in both fundamental physiology and in human diseases, mainly cancers. An emerging pattern suggests that BORG family members cancer‐promoting properties are related to their ability to regulate the cytoskeleton, with many impacting the organization of acto‐myosin stress fibers. This is consistent with the broader literature indicating that BORG family members are regulators of both the septin and actin cytoskeleton networks. The exact mechanism through which BORGs modify the cytoskeleton is not clear, but we consider here a few data‐supported and speculative possibilities. Finally, we delve into how the Rho GTPase Cdc42 modifies BORG function in cells. This remains open‐ended as Cdc42's effects on BORGs appear cell type‐ and cell state‐dependent. Collectively, these data point to the importance of the BORG family and suggest broader themes in their function and regulation.
Hematological cancers are among the most common cancers in adults and in children. Despite significant improvements in therapies, many patients still succumb to the disease, therefore, novel therapies are needed. The Wiskott-Aldrich syndrome protein (WASp) family proteins regulate actin assembly in conjunction with the Arp2/3 complex, a ubiquitous nucleation factor. WASp is expressed exclusively in hematopoietic cells and exists in two allosteric conformations, auto-inhibited and active conformations. Here, we describe the development of EG-011, a first-in-class small molecule activator of the WASp auto-inhibited form. EG-011 possesses in vitro and in vivo anti-tumor activity as single agent in lymphoma, leukemia and multiple myeloma, including models of secondary resistance to PI3K, BTK and proteasome inhibitors. The in vitro activity was confirmed in a lymphoma xenograft. Actin polymerization induced by EG-011 was demonstrated with multiple techniques. Transcriptome analysis highlighted homology with drugs inducing actin polymerization.
Background. Novel therapeutic targets are needed to improve the outcome of individuals with cancer. EG-011 is a small molecule with in vitro and in vivo anti-tumor activity in lymphoma and acute leukemias, but, at least so far, no activity in solid tumor models (Gaudio et al AACR 2019). Since no information was available on its target, we have now performed experiments showing that EG-011 targets WASp. Methods. Target identification: kinase screens with DiscoverX KINOMEscan, ProQinase Wildtype-Profiler; thermal proteomic profiling. Target validation: pyrene actin polymerization assay. In vitro drug treatment of cell lines followed for changes in actin filaments (F-actin) by confocal imaging with Alexa Fluor 488 Phalloidin. Results. Extensive kinome screens excluded kinases as targets of EG-011. We then applied thermal proteomic profiling to identify new protein targets interacting with EG-011. Over 3,300 proteins from the soluble proteome from the EG-011 sensitive mantle cell lymphoma cell line REC1 were analyzed and 48 possible protein targets were initially identified. Among the proteins undergoing a thermal shift, WASp was among the most highly destabilized by EG-011. Due to the pattern of expression of WASp, compatible with the anti-tumor activity observed only in hematological cancers (Gaudio et al AACR 2019), we performed further experiments to confirm the possibility that EG-011 targets WASp. One of the main functions of WASp is the regulation of actin filaments formation. Pyrene actin polymerization assays demonstrated that EG-011 activated the auto-inhibited form of WASP with strong actin polymerization. Further confirmation was obtained using confocal imaging of cell lines exposed to DMSO or EG-011 (500 nM, 5 μM) and stained for F-actin. An increase in actine polymerization was seen in EG-011 sensitive (VL51) and not in resistant (Z138) cell lines at 4, 8 and 24h with both concentrations. Conclusions. These data demonstrate that EG-011 is the “first-in-class” activator of the auto-inhibited form of WASp with selective anti-tumor activity in lymphomas. Citation Format: Filippo Spriano, Laura Barnabei, Ana Maria Carrasco Del Amor, Meagan R. Tomasso, Chiara Tarantelli, Eugenia Riveiro, Natalina Pazzi, Shae B. Padrick, Susana Cristobal, Franco Cavalli, Eugenio Gaudio, Francesco Bertoni. EG-011 is a first-in-class Wiskott-Aldrich syndrome protein (WASp) activator with anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1817.
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