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The Rho GEF Trio is transported by microtubule plus-ends and involved in focal adhesion formation in migrating neural crest cells
Directed cell migration depends on cytoskeletal rearrangements, protrusion formation, cell contraction and focal adhesion turnover. These processes are regulated by spatiotemporal fine-tuning of Rho GTPase activity. The Rho guanine nucleotide exchange factor (GEF) Trio is well suited to control R...
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| Format: | Doctoral Thesis |
| Language: | English |
| Published: |
Philipps-Universität Marburg
2023
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| Online Access: | PDF Full Text |
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| Summary: | Directed cell migration depends on cytoskeletal rearrangements, protrusion formation, cell
contraction and focal adhesion turnover. These processes are regulated by spatiotemporal
fine-tuning of Rho GTPase activity. The Rho guanine nucleotide exchange factor (GEF) Trio
is well suited to control Rho GTPase activity by merging two catalytic GEF domains, allowing
control over Rac1 and RhoA within a single protein. However, strict spatiotemporal control of
the activity of the Trio GEF domains at the cellular level is necessary. We recently
demonstrated that Trio is required for Xenopus neural crest (NC) cell protrusion formation and
migration. Here, we examine the dynamic localization of Trio and its impact on Trio's role in
NC cell migration. Live-cell imaging revealed that the Trio GEF2 domain co-localizes with EB3
at microtubule plus-ends. Microtubule trafficking of Trio appears to be important for its function,
as a mutant GEF2 construct lacking the SxIP amino acid motif responsible for microtubule
plus-end binding, was unable to rescue the Trio loss-of-function induced NC migration defects
in vivo and in vitro. In addition, our analysis of microtubule dynamics in migrating neural crest
cells revealed that Trio knockdown results in the stabilization of microtubules at cell-cell
contacts, while destabilizing them at the leading edge compared to the control. Furthermore,
our findings indicate that Trio is involved in focal adhesion dynamics, as analyzed by live-cell
imaging of focal adhesion assembly and disassembly. Our data suggest that Trio is transported
by microtubules to specific subcellular locations, where it has distinct functions in controlling
microtubule stability, protrusion formation and adhesive functions during directed NC cell
migration. Furthermore, TRIO gene mutations have been shown to cause neurodevelopmental
disorders and facial dysmorphisms in patients, possibly by inhibiting the migration of neural
crest cells. Similar clinical features were observed in individuals with mutations in the MAPRE2
and TUBB genes. We successfully induced Mapre2 and Tubb loss-of-function in Xenopus
embryos by injecting specific translation-blocking morpholinos and demonstrate, comparable
to patient data, that this knockdown results in NC migration defects and craniofacial
malformations. These experiments will serve as a starting point to analyze whether Trio,
Mapre2 and Tubb operate within the same signaling pathways to regulate microtubule
dynamics, focal adhesion turnover and, thereby, cell motility. |
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| DOI: | 10.17192/z2024.0075 |