RESUMO
Dynamic interactions between RhoA and Rac1, members of the Rho small GTPase family, play a vital role in the control of cell migration. Using predictive mathematical modeling, mass spectrometry-based quantitation of network components, and experimental validation in MDA-MB-231 mesenchymal breast cancer cells, we show that a network containing Rac1, RhoA, and PAK family kinases can produce bistable, switch-like responses to a graded PAK inhibition. Using a small chemical inhibitor of PAK, we demonstrate that cellular RhoA and Rac1 activation levels respond in a history-dependent, bistable manner to PAK inhibition. Consequently, we show that downstream signaling, actin dynamics, and cell migration also behave in a bistable fashion, displaying switches and hysteresis in response to PAK inhibition. Our results demonstrate that PAK is a critical component in the Rac1-RhoA inhibitory crosstalk that governs bistable GTPase activity, cell morphology, and cell migration switches.
Assuntos
Citoesqueleto de Actina , Actinas , Linhagem Celular Tumoral , Movimento Celular , Citoesqueleto , Humanos , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP , Proteína rhoA de Ligação ao GTPRESUMO
Cilia are microtubule-based cell appendages, serving motility, chemo-/mechano-/photo- sensation, and developmental signaling functions. Cilia are comprised of distinct structural and functional subregions including the basal body, transition zone (TZ) and inversin (Inv) compartments, and defects in this organelle are associated with an expanding spectrum of inherited disorders including Bardet-Biedl syndrome (BBS), Meckel-Gruber Syndrome (MKS), Joubert Syndrome (JS) and Nephronophthisis (NPHP). Despite major advances in understanding ciliary trafficking pathways such as intraflagellar transport (IFT), how proteins are transported to subciliary membranes remains poorly understood. Using Caenorhabditis elegans and mammalian cells, we investigated the transport mechanisms underlying compartmentalization of JS-associated ARL13B/ARL-13, which we previously found is restricted at proximal ciliary membranes. We now show evolutionary conservation of ARL13B/ARL-13 localisation to an Inv-like subciliary membrane compartment, excluding the TZ, in many C. elegans ciliated neurons and in a subset of mammalian ciliary subtypes. Compartmentalisation of C. elegans ARL-13 requires a C-terminal RVVP motif and membrane anchoring to prevent distal cilium and nuclear targeting, respectively. Quantitative imaging in more than 20 mutants revealed differential contributions for IFT and ciliopathy modules in defining the ARL-13 compartment; IFT-A/B, IFT-dynein and BBS genes prevent ARL-13 accumulation at periciliary membranes, whereas MKS/NPHP modules additionally inhibit ARL-13 association with TZ membranes. Furthermore, in vivo FRAP analyses revealed distinct roles for IFT and MKS/NPHP genes in regulating a TZ barrier to ARL-13 diffusion, and intraciliary ARL-13 diffusion. Finally, C. elegans ARL-13 undergoes IFT-like motility and quantitative protein complex analysis of human ARL13B identified functional associations with IFT-B complexes, mapped to IFT46 and IFT74 interactions. Together, these findings reveal distinct requirements for sequence motifs, IFT and ciliopathy modules in defining an ARL-13 subciliary membrane compartment. We conclude that MKS/NPHP modules comprise a TZ barrier to ARL-13 diffusion, whereas IFT genes predominantly facilitate ARL-13 ciliary entry and/or retention via active transport mechanisms.
Assuntos
Fatores de Ribosilação do ADP/genética , Caenorhabditis elegans/genética , Doenças Cerebelares/genética , Cílios/genética , Anormalidades do Olho/genética , Doenças Renais Císticas/genética , Retina/anormalidades , Fatores de Ribosilação do ADP/metabolismo , Anormalidades Múltiplas , Animais , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patologia , Transporte Biológico Ativo/genética , Caenorhabditis elegans/metabolismo , Doenças Cerebelares/patologia , Cerebelo/anormalidades , Cílios/metabolismo , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/patologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Encefalocele/genética , Encefalocele/patologia , Anormalidades do Olho/patologia , Humanos , Doenças Renais Císticas/patologia , Membranas/metabolismo , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Retina/patologia , Retinose Pigmentar , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and related ciliopathies present with overlapping phenotypes and display considerable allelism between at least twelve different genes of largely unexplained function. We demonstrate that the conserved C. elegans B9 domain (MKS-1, MKSR-1, and MKSR-2), MKS-3/TMEM67, MKS-5/RPGRIP1L, MKS-6/CC2D2A, NPHP-1, and NPHP-4 proteins exhibit essential, collective functions at the transition zone (TZ), an underappreciated region at the base of all cilia characterized by Y-shaped assemblages that link axoneme microtubules to surrounding membrane. These TZ proteins functionally interact as members of two distinct modules, which together contribute to an early ciliogenic event. Specifically, MKS/MKSR/NPHP proteins establish basal body/TZ membrane attachments before or coinciding with intraflagellar transport-dependent axoneme extension and subsequently restrict accumulation of nonciliary components within the ciliary compartment. Together, our findings uncover a unified role for eight TZ-localized proteins in basal body anchoring and establishing a ciliary gate during ciliogenesis, and suggest that disrupting ciliary gate function contributes to phenotypic features of the MKS/NPHP disease spectrum.