Introduction
Cell division control protein 42 homolog (Cdc42), also known as cell division cycle 42, belongs to the Rho family of small GTPases and is notable as a central signaling hub that coordinates a myriad of fundamental cellular processes.1 Extensive studies have established key roles in regulating cytoskeletal dynamics and other downstream pathways to control cell shape and motility, differentiation, and cell cycle progression.2,3
Structurally, Cdc42 resembles other Rho GTPases and contains the two essential switch domains that mediate interactions with upstream regulators and downstream effectors, as well as enabling nucleotide binding.4 The protein is ubiquitously expressed2,5 and, in common with other Rho GTPases, relies on membrane localization to effect downstream signaling.2 This is achieved by posttranslational lipidation at Cys188, located in the C-terminal domain.1,6
Cdc42 is particularly known for its unique role in controlling cell polarity and in modulating polarized cell functions, including the proper formation of epithelial tight and adherens junctions.6-8 Thus, it is essential for correct morphogenesis of organs such as the heart, lungs, pancreas, and kidneys during early development.9,10 This newsletter summarizes the roles of Cdc42 in these processes and describes recently identified developmental and autoinflammatory disorders resulting from mutations in key functional domains.
Cdc42 Functions in Epithelial Polarization and Morphogenesis
Apical–basal polarity is a fundamental organizing principle of epithelial cells (Figure 1) and relies on a range of conserved factors including the PAR complex, a key regulator of cell polarity.11 Early studies defined its components as PAR-3, PAR-6, and “atypical protein kinase C” (aPKC), referring to the PKCζ (PRKCZ) and PKCι (PRKCI) isoforms. This complex is typically found at the apical membrane in polarized epithelial cells and is also known to facilitate formation of tight and adherens junctions.9 Work on embryoid bodies derived from Cdc42+/− and Cdc42−/− murine embryonic stem cells9 identified Cdc42 as an apparent regulator of the PAR complex, mediating epithelial tissue morphogenesis by maintaining its apical polarity and contributing to junction maturation.12 Later research developed a refined model where an inactive PAR 3/PAR 6/aPKC complex is trafficked to the apical region by actomyosin cortical flows, then membrane-bound activated Cdc42 displaces PAR 3 to form the active Cdc42/PAR 6/aPKC complex.13,14
Junction maturation is seemingly controlled by PAK4, a known downstream effector of Cdc42.15 Additionally, Cdc42 has key roles in epithelial morphogenesis through controlling apical actin organization16 and spindle orientation during cell division, ensuring the mitotic spindle is aligned to enable correct positioning of apical surfaces.16,17 By regulating these processes, Cdc42 is critical for proper development of multiple organs and tissues, as has been demonstrated in several studies using tissue-specific Cdc42-knockout mice based on the Cre/loxP system.18
Tubulogenesis, Cdc42 is important in forming the glandular structure of the pancreas.19 During murine embryonic development, the process starts with microlumen formation throughout the developing epithelium followed by fusion and arrangement into interconnected tubes. This relies specifically on Cdc42 for establishment and maintenance of multicellular apical polarization, through the PAR complex and other pathways. Tubulogenesis is equally critical in the developing kidney, where Cdc42 controls iterative branching at the ureteric bud to generate the renal collecting duct system. Deleting Cdc42 in the ureteric bud of developing mouse embryos caused severe branching defects, leading to smaller kidneys with histological features of end-stage renal disease.3 This was attributed to dysregulation of both the PAR complex and actin organization via N-WASP, a well-known downstream effector of Cdc42.
Podocyte function, deletion of Cdc42 induced congenital nephropathy and renal failure due to abnormal morphology of the slit diaphragm, an essential structure of the glomerular filtration barrier that is formed from interconnected foot processes of podocytes.32 Other studies identified an unexpected role for Cdc42 in nephrogenesis, where it controls nuclear localization of the YAP1 transcription factor to regulate cell fate and morphogenesis.33 Interestingly, Cdc42/YAP1 signaling also appears to protect against apoptosis in mature podocytes.34
Ventricular development, the embryonic heart also requires Cdc42, since cardiomyocyte-specific knockout caused ventricular abnormalities due to reduced proliferation and cell–cell adhesion.20 Disorganized sarcomeres were observed due to deficient formation of adherens junctions that are essential for mechanical coupling between cardiomyocytes. Furthermore, Cdc42 is required for proper formation of the epicardium, in part through polarization of proepicardial cells but also by controlling membrane trafficking of the FGFR1 receptor to regulate cellular proliferation via FGF2 signaling.21
Above: Model for transition from Par-3- to Cdc42-bound Par complex. Adapted from Vargas, Prehoda13.
Cdc42 Isoforms and Neurodevelopment
Depletion of Cdc42 also causes serious defects in CNS development,22 yet the underlying mechanisms remained poorly understood for a long time. This is partly because two Cdc42 isoforms are expressed in the brain, which vary in their final exon through alternative splicing and therefore have different C-terminal sequences. While both isoforms have the same interactome in terms of known regulatory factors and effectors,6 differential posttranslational lipidation controls their subcellular localization to regulate distinct cellular functions. Specifically, the ubiquitously expressed form (Cdc42u) contains a canonical prenylation site at Cys188, whereas the brain-specific variant (Cdc42b) is preferentially palmitoylated at its additional cysteine residue, Cys189.1,23
Thus, prenylated and palmitoylated forms of Cdc42 are present in brain and current evidence suggests they play different roles in neurogenesis.5 Through opposing effects on mTORC1 activity, Cdc42u mediates initial formation of neural progenitors while Cdc42b promotes terminal differentiation into neurons. Furthermore, enrichment of Cdc42b has been reported in neuronal somata and dendritic spines,1 and a role in N WASP-mediated endocytosis is suggested in neural precursors,6 consistent with findings that the brain-specific isoform mediates EGFR degradation to help trigger neuronal differentiation.22
Pathogenic Cdc42 Mutations
In 2015, Takenouchi–Kosaki syndrome was first documented as a rare autosomal dominant disorder associated with heterozygous CDC42 mutations. Clinical features include facial abnormalities, developmental delay, and multisystemic anomalies such as lymphedema and macrothrombocytopenia.7,24 The predominant mutation, Y64C, affects the essential switch II region of Cdc42, leading to its increased activation and insensitivity toward GEFs and GAPs.24,25
Later, a second group of CDC42-associated hematopoietic and autoinflammatory disorders was reported due to mutations in the C-terminal region, most notably R186C.26 This mutation results in aberrant palmitoylation of the pathogenic variant, causing mislocalization to the Golgi apparatus and driving pyrin inflammasome hyperactivation.7,27 Recent work has suggested that R186C and other known C terminal mutations all cause NF-κB hyperactivation, unlike the Y64C variant.28 It is hoped that such studies will pave the way for targeted therapeutic interventions.
Outstanding Questions
While existing studies have uncovered many aspects of Cdc42 function, important questions still remain. A detailed understanding of the two Cdc42 isoforms and their physiological roles is currently lacking,1 not least because many studies have failed to discriminate between these variants. Interestingly, Cdc42b may have functional roles beyond the brain as it has been detected at low levels in multiple non-neuronal cell lines including MDCKII, NIH3T3, and CHO cells.23
Moreover, while several regulatory GEFs, GAPs, and GDIs are known for Cdc42,29 considerable gaps remain in our knowledge of these complex networks and how they orchestrate the known functions of Cdc42.30 In particular, our understanding of Cdc42 spatiotemporal activation in polarized epithelial cells is incomplete,15 and much is still unknown about the functioning of polarity proteins during mammalian development.14 Intriguing recent findings also hint at an additional layer of complexity beyond canonical regulation. The long noncoding RNA TINCR encodes an 87-amino-acid microprotein that is highly expressed in epithelia and induces epithelial differentiation by promoting Cdc42 activation.31 Thus, many opportunities remain to explore the diverse roles of Cdc42 in development and beyond.
References
1. Wirth A, Ponimaskin E. Lipidation of small GTPase Cdc42 as regulator of its physiological and pathophysiological functions. Front Physiol. 2023;13:1088840. https://doi.org/10.3389/fphys.2022.1088840.
2. Murphy NP, binti Ahmad Mokhtar AM, Mott HR, Owen D. Molecular subversion of Cdc42 signalling in cancer. Biochem Soc Trans. 2021;49(3):1425–1442. https://doi.org/10.1042/bst20200557.
3. Elias BC, Das A, Parekh DV, et al. Cdc42 regulates epithelial cell polarity and cytoskeletal function during kidney tubule development. J Cell Sci. 2015;128(23):4293–4305. https://doi.org/10.1242/jcs.164509.
4. Toma-Fukai S, Shimizu T. Structural insights into the regulation mechanism of small GTPases by GEFs. Molecules. 2019;24(18):3308. https://doi.org/10.3390/molecules24183308.
5. Endo M, Druso JE, Cerione RA. The two splice variant forms of Cdc42 exert distinct and essential functions in neurogenesis. J Biol Chem. 2020;295(14):4498–4512. https://doi.org/10.1074/jbc.ra119.011837.
6. Ravichandran Y, Hänisch J, Murray K, et al. The distinct localization of CDC42 isoforms is responsible for their specific functions during migration. J Cell Biol. 2024;223(3):e202004092. https://doi.org/10.1083/jcb.202004092.
7. Su HC, Orange JS. The growing spectrum of human diseases caused by inherited CDC42 mutations. J Clin Immunol. 2020;40(4):551–553. https://doi.org/10.1007/s10875-020-00785-8.
8. Fu J, Liu B, Zhang H, et al. The role of cell division control protein 42 in tumor and non-tumor diseases: A systematic review. J Cancer. 2022;13(3):800–814. https://doi.org/10.7150/jca.65415.
9. Wu X, Li S, Chrostek-Grashoff A, Czuchra A, Meyer H, Yurchenco PD, Brakebusch C. Cdc42 is crucial for the establishment of epithelial polarity during early mammalian development. Dev Dyn. 2007;236(10):2767–2778. https://doi.org/10.1002/dvdy.21309.
10. Wan H, Liu C, Wert SE, Xu W, Liao Y, Zheng Y, Whitsett JA. CDC42 is required for structural patterning of the lung during development. Dev Biol. 2013;374(1):46–57. https://doi.org/10.1016/j.ydbio.2012.11.030.
11. Chen J, Zhang M. The Par3/Par6/aPKC complex and epithelial cell polarity. Exp Cell Res. 2013;319(10):1357–1364. https://doi.org/10.1016/j.yexcr.2013.03.021.
12. Melendez J, Grogg M, Zheng Y. Signaling role of Cdc42 in regulating mammalian physiology. J Biol Chem. 2011;286(4):2375–2381. https://doi.org/10.1074/jbc.r110.200329.
13. Vargas E, Prehoda KE. Negative cooperativity underlies dynamic assembly of the Par complex regulators Cdc42 and Par-3. J Biol Chem. 2023;299(1):102749. https://doi.org/10.1016/j.jbc.2022.102749.
14. Deutz LN, Sarıkaya S, Dickinson DJ. Membrane extraction in native lipid nanodiscs reveals dynamic regulation of Cdc42 complexes during cell polarization. Biophys J. Published online November 23, 2023. https://doi.org/10.1016/j.bpj.2023.11.021.
15. Pichaud F, Walther RF, Nunes de Almeida F. Regulation of Cdc42 and its effectors in epithelial morphogenesis. J Cell Sci. 2019;132(10):jcs217869. https://doi.org/10.1242/jcs.217869.
16. Buckley CE, St Johnston D. Apical–basal polarity and the control of epithelial form and function. Nat Rev Mol Cell Biol. 2022;23(8):559–577. https://doi.org/10.1038/s41580-022-00465-y.
17. Jaffe AB, Kaji N, Durgan J, Hall A. Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis. J Cell Biol. 2008;183(4):625–633.
18. McLellan MA, Rosenthal NA, Pinto AR. Cre-loxP-mediated recombination: General principles and experimental considerations. Curr Protoc Mouse Biol. 2017;7(1):1–12. https://doi.org/10.1002/cpmo.22.
19. Kesavan G, Sand FW, Greiner TU, et al. Cdc42-mediated tubulogenesis controls cell specification. Cell. 2009;139(4):791–801. https://doi.org/10.1016/j.cell.2009.08.049.
20. Li J, Liu Y, Jin Y, et al. Essential role of Cdc42 in cardiomyocyte proliferation and cell–cell adhesion during heart development. Dev Biol. 2017;421(2):271–283. https://doi.org/10.1016/j.ydbio.2016.12.012.
21. Li J, Miao L, Zhao C, et al. CDC42 is required for epicardial and pro-epicardial development by mediating FGF receptor trafficking to the plasma membrane. Development. 2017;144(9):1635–1647. https://doi.org/10.1242/dev.147173.
22. Endo M, Cerione RA. The brain-specific splice variant of the CDC42 GTPase works together with the kinase ACK to downregulate the EGF receptor in promoting neurogenesis. J Biol Chem. 2022;298(11):102564. https://doi.org/10.1016/j.jbc.2022.102564.
23. Wirth A, Chen-Wacker C, Wu Y-W, Gorinski N, Filippov MA, Pandey G, Ponimaskin E. Dual lipidation of the brain-specific Cdc42 isoform regulates its functional properties. Biochem J. 2013;456(3):311–322. https://doi.org/10.1042/bj20130788.
24. Martinelli S, Krumbach OHF, Pantaleoni F, et al. Functional dysregulation of CDC42 causes diverse developmental phenotypes. Am J Hum Genet. 2018;102(2):309–320. https://doi.org/10.1016/j.ajhg.2017.12.015.
25. Hamada N, Ito H, Shibukawa Y, Morishita R, Iwamoto I, Okamoto N, Nagata K-I. Neuropathophysiological significance of the c.1449T>C/p.(Tyr64Cys) mutation in the CDC42 gene responsible for Takenouchi–Kosaki syndrome. Biochem Biophys Res Commun. 2020;529(4):1033–1037. https://doi.org/10.1016/j.bbrc.2020.06.104.
26. Coppola S, Insalaco A, Zara E, et al. Mutations at the C-terminus of CDC42 cause distinct hematopoietic and autoinflammatory disorders. J Allergy Clin Immunol. 2022;150(1):223–228. https://doi.org/10.1016/j.jaci.2022.01.024.
27. Nishitani-Isa M, Mukai K, Honda Y, et al. Trapping of CDC42 C-terminal variants in the Golgi drives pyrin inflammasome hyperactivation. J Exp Med. 2022;219(6):e20211889. https://doi.org/10.1084/jem.20211889.
28. Iannuzzo A, Mertz P, Delafontaine S, et al. C-terminal CDC42 variants in autoinflammatory patients specifically trigger actin defects and NF-κB hyperactivation. bioRxiv. Preprint posted online June 30, 2024. https://doi.org/10.1101/2024.06.26.600829.
29. Arias-Romero LE, Chernoff J. Targeting Cdc42 in cancer. Expert Opin Ther Targets. 2013;17(11):1263–1273. https://doi.org/10.1517/14728222.2013.828037.
30. Dahmene M, Quirion L, Laurin M. High throughput strategies aimed at closing the GAP in our knowledge of Rho GTPase signaling. Cells. 2020;9(6):1430. https://doi.org/10.3390/cells9061430.
31. Boix O, Martinez M, Vidal S, et al. pTINCR microprotein promotes epithelial differentiation and suppresses tumor growth through CDC42 SUMOylation and activation. Nat Commun. 2022;13(1):6840. https://doi.org/10.1038/s41467-022-34529-6.
32. Scott RP, Hawley SP, Ruston J, Du J, Brakebusch C, Jones N, Pawson T. Podocyte-specific loss of Cdc42 leads to congenital nephropathy. J Am Soc Nephrol. 2012;23(7):1149–1154. https://doi.org/10.1681/asn.2011121206.
33. Reginensi A, Scott RP, Gregorieff A, et al. Yap- and Cdc42-dependent nephrogenesis and morphogenesis during mouse kidney development. PLoS Genet. 2013;9(3):e1003380. https://doi.org/10.1371/journal.pgen.1003380.
34. Huang Z, Zhang L, Chen Y, et al. Cdc42 deficiency induces podocyte apoptosis by inhibiting the Nwasp/stress fibers/YAP pathway. Cell Death Dis. 2016;7(3):e2142. https://doi.org/10.1038/cddis.2016.51.