The Crk adapter protein is essential forDrosophilaembryogenesis, where it regulates multiple actin-dependent morphogenic events

Abstract

Small Src homology domain 2 (SH2) and 3 (SH3) adapter proteins regulate cell fate and behavior by mediating interactions between cell surface receptors and downstream signaling effectors in many signal transduction pathways. The CT10 regulator of kinase (Crk) family has tissue-specific roles in phagocytosis, cell migration, and neuronal development and mediates oncogenic signaling in pathways like that of Abelson kinase. However, redundancy among the two mammalian family members and the position of the Drosophila gene on the fourth chromosome precluded assessment of Crk’s full role in embryogenesis. We circumvented these limitations with short hairpin RNA and CRISPR technology to assess Crk’s function in Drosophila morphogenesis. We found that Crk is essential beginning in the first few hours of development, where it ensures accurate mitosis by regulating orchestrated dynamics of the actin cytoskeleton to keep mitotic spindles in syncytial embryos from colliding. In this role, it positively regulates cortical localization of the actin-related protein 2/3 complex (Arp2/3), its regulator suppressor of cAMP receptor (SCAR), and filamentous actin to actin caps and pseudocleavage furrows. Crk loss leads to the loss of nuclei and formation of multinucleate cells. We also found roles for Crk in embryonic wound healing and in axon patterning in the nervous system, where it localizes to the axons and midline glia. Thus, Crk regulates diverse events in embryogenesis that require orchestrated cytoskeletal dynamics.

Abelson kinase’s intrinsically disordered region plays essential roles in protein function and protein stability

“…Other mutants, including those that disrupt syncytial development and cellularization in different ways, as is seen in embryos mutant for the septin peanut , lead to a similarly disrupted cuticle phenotype [ 57 ]. Intriguingly, embryos maternally and zygotically mutant for the adapter protein Crk, which in mammals can bind the Abl PXXP motif and is thought to act as an Abl regulator or effector [ 11 , 12 ], also have strong defects in syncytial development and cellularization, leading to strong disruption of epithelial integrity [ 45 ], as we observed here. Crk regulates actin dynamics in the early Drosophila embryo by recruiting SCAR to the cortex [ 45 ], and the PXXP motif within Abl’s IDR can bind proteins in the WAVE regulatory complex [ 46 ], of which Scar is a part.…”

“…Surprisingly, however, this motif was extremely important, as its deletion reduced Abl function more than what was caused by loss of kinase activity or even loss of both kinase activity and the FABD. Subsequent analyses support the idea that this motif acts by interactions with the adapter protein Crk [ 45 ] and with the actin-regulatory WAVE complex [ 46 ]. The fine-grained dissections of the IDR by Cheong and VanBerkum suggest other regions of the IDR may have more subtle roles in axon guidance.…”

Crk adaptor proteins are necessary for the development of the zebrafish retina

“…Their expression throughout the retinal layers later in eye development would enable them to engage in a diversity of signaling pathways, directing retinal cells toward migration or differentiation as has been shown previously in other tissues. 6,15,16,[35][36][37][38][39] Cell fate would be dependent on the differential localization and abundance of phosphotyrosine-containing proteins to which Crk adaptors could bind via their SH2 domains, as well as the relative concentration of Crk family SH3-binding effector proteins in a given cell. We previously identified 101 CRKL-SH3-binding partners in embryonic murine brain extract, 40 and thus, it is anticipated that the combinatorial outcomes of Crk adaptordependent signal transduction would be numerous and dependent on kinetic-based competition of effectors.…”

“…In Drosophilia, where there is only one CRK family member, Crk was found to be important for early ectoderm cell divisions through coordination of mitotic spindle actin dynamics, revealing a role in axon guidance and wound healing later in development. 15 In zebrafish, knockdown via morpholinos and CRISPRi demonstrated roles for both Crk and Crkl in myocyte fusion and podocyte morphology 16,17 although no nervous system phenotypes due to loss of Crk adaptors in zebrafish have been described to date. In the mouse model, targeted disruption of CRK leads to embryos with cardiac defects, craniofacial abnormalities, and hemorrhagic edema in smooth muscle.…”

Bitesize bundles F-actin and influences actin remodeling in syncytialDrosophilaembryo development

“…Pseudo-cleavage furrows act as a barrier between dividing spindles and are essential for proper nuclear divisions. Perturbing pseudo-cleavage furrows with drugs or depleting actin-related proteins often results in spindle collisions (Afshar et al, 2000;Callaini et al, 1992;Spracklen et al, 2019;Sullivan et al, 1993;Webb et al, 2009;Zallen et al, 2002).…”