Mechanistic Insight into the Microtubule and Actin Cytoskeleton Coupling through Dynein-Dependent RhoGEF Inhibition
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Actin-based stress fiber formation is coupled to microtubule depolymerization through the local activation of RhoA. While the RhoGEF Lfc has been implicated in this cytoskeleton coupling process, it has remained elusive how Lfc is recruited to microtubules and how microtubule recruitment moderates Lfc activity. Here, we demonstrate that the dynein light chain protein Tctex-1 is required for localization of Lfc to microtubules. Lfc residues 139-161 interact with Tctex-1 at a site distinct from the cleft that binds dynein intermediate chain. An NMR-based GEF assay revealed that interaction with Tctex-1 represses Lfc nucleotide exchange activity in an indirect manner that requires both polymerized microtubules and phosphorylation of S885 by PKA. We show that inhibition of Lfc by Tctex-1 is dynein dependent. These studies demonstrate a pivotal role of Tctex-1 as a negative regulator of actin filament organization through its control of Lfc in the crosstalk between microtubule and actin cytoskeletons.
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GEF-H1 is required for LPA/thrombin-induced stress fibre formation and focal adhesion formation following stimulation with tumour necrosis factor-a, LPA or thrombin.
“GEF-H1 is required for LPA/thrombin-induced stress fibre formation, and focal adhesion formation following stimulation with tumour necrosis factor-a, LPA or thrombin 2,3,6 .”
From Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1 (2014) · cites this paper as [6]
The Results and Discussion report that Lfc (GEF-H1) is required for LPA-induced stress fibres and focal adhesions in mouse embryonic fibroblasts, and the Discussion describes a role in LPA/thrombin-induced stress fibre formation. The paper also examines focal adhesions after thrombin stimulation, but the passages found do not address focal adhesion formation after tumour necrosis factor-α stimulation (searched: tumour necrosis factor, TNF-α). (LLM, conf=0.87)
Experimental — accuracy not yet measuredGEF-H1 activation results from disruption of the GEF-H1:dynein complex by G-protein subunits in the absence of microtubule depolymerization.
“Our results suggest a model where GEF-H1 activation results from disruption of the GEF-H1:dynein complex by G-protein subunits in the absence of microtubule depolymerization 6,7 .”
From Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1 (2014) · cites this paper as [6]
The Discussion identifies the dynein light chain Tctex-1 as anchoring Lfc (GEF-H1) to polymerized microtubules, and reports that Tctex-1 represses its exchange activity. It does not show G-protein subunits disrupting that complex or activating GEF-H1 without microtubule depolymerization; searches for those mechanisms found no supporting passage. (LLM, conf=0.85)
Experimental — accuracy not yet measuredNocodazole-induced microtubule depolymerization potently activates GEF-H1.
“Nocodozole-induced microtubule depolymerization potently activates GEF-H1 (refs 5-7), thus GEF-H1 activity during mitosis may be a consequence of the change in microtubule dynamics during the reorganization of stable interphase microtubules into the mitotic spindle 36 .”
From Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1 (2014) · cites this paper as [6]
In “Polymerized Microtubules Are Required for Tctex-1-Mediated Inhibition of Lfc” (page not provided), the paper reports that nocodazole increased Lfc/GEF-H1 exchange activity 1.5-fold in lysates of Lfc-overexpressing cells. This supports activation after microtubule depolymerization, but does not establish that the activation was potent or that the measured effect applies beyond those cells. (LLM, conf=0.84)
Experimental — accuracy not yet measuredMicrotubule-dependent inhibition of GEF-H1 requires the assembly of a protein complex including Tctex-1, dynein intermediate chain (DIC) and 14-3-3 proteins.
“We have recently shown that microtubule-dependent inhibition of GEF-H1 requires the assembly of a protein complex including Tctex-1, dynein intermediate chain (DIC) and 14-3-3 proteins 6 .”
From Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1 (2014) · cites this paper as [6]
The Discussion (page unavailable) says that Lfc inhibition depends on a complex containing Tctex-1 and dynein intermediate chain, and that intact microtubules are required. It also reports that phosphorylation creates a 14-3-3 binding site that inhibits Lfc activity, but does not establish that 14-3-3 proteins are required members of the Tctex-1–DIC complex. (LLM, conf=0.86)
Experimental — accuracy not yet measuredThe dynein light-chain Tctex-1 tethers GEF-H1 to polymerized microtubules.
“We have shown that the dynein light-chain Tctex-1 tethers GEF-H1 to polymerized microtubules 6 , where it is poised to undergo D-AKAP-1dependent phosphorylation of Ser885 by PKA.”
From Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1 (2014) · cites this paper as [6]
Co-expression of the Gbg subunits relieved Tctex-1-mediated inhibition of GEF-H1 exchange activity.
“As previously reported, Tctex-1 potently inhibited the exchange activity of full-length GEF-H1 (refs 6,17), whereas co-expression of the Gbg subunits relieved this inhibitory effect (Fig. 2e).”
From Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1 (2014) · cites this paper as [6]
Recombinant full-length GEF-H1 protein expressed and purified from insect cells bound to recombinant Ga13, indicating that active Ga can bind directly to GEF-H1 in vitro.
“Recombinant full-length GEF-H1 protein expressed and purified from insect cells 6 bound to recombinant Ga 13 , indicating that active Ga can bind directly to GEF-H1 in vitro (Supplementary Fig. 4a).”
From Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1 (2014) · cites this paper as [6]
The passage on full-length Lfc/GEF-H1 (labeled Abstract in our copy; page unavailable) describes assays in HEK293T cell lysates and regulation by Tctex-1, not binding to Gα13. Searches for Gα13 binding, purified protein from insect cells, and Supplementary Fig. 4a found no passage establishing the claimed direct interaction. (LLM, conf=0.76)
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Cites
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- Authors
- Meiri, D., Marshall, C. B., Greeve, M. A., Kim, B., Balan, M., Suarez, F., Bakal, C., Wu, C., LaRose, J., Fine, N., Ikura, M., & Rottapel, R.
- Affiliations
- University of Toronto (CA)
- St. Michael's Hospital (CA)
- Corresponding author
- Ikura, Mitsuhiko
- Rottapel, Robert
- ORCID
- Venue
- Molecular Cell
- Publisher
- Elsevier BV
- Published
- 2012-03
- Volume
- 45
- Issue
- 5
- Pages
- 642-655
- ISSN
- 1097-2765 (ISSN-L)
- OpenAlex
- W2142562240
- Cited by
- 98
- References
- 1
- Citations by year
- 2026: 5 · 2025: 7 · 2024: 4 · 2023: 3 · 2022: 5 · 2021: 8 · 2020: 9 · 2019: 5 · 2018: 10 · 2017: 11 · 2016: 5 · 2015: 6 · 2014: 9 · 2013: 7 · 2012: 4
- FWCI
- 3.67
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.elsevier.com/tdm/userlicense/1.0/ (tdm)
- www.elsevier.com/legal/tdmrep-license (tdm)
- www.elsevier.com/open-access/userlicense/1.0/ (vor, 539 d)
- doi.org/10.15223/policy-017 (stm-asf)
- doi.org/10.15223/policy-037 (stm-asf)
- doi.org/10.15223/policy-012 (stm-asf)
- doi.org/10.15223/policy-029 (stm-asf)
- doi.org/10.15223/policy-004 (stm-asf)
- Locations
- Molecular Cell (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Canadian Institutes of Health Research (102745_1)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular Mechanics and Interactions
- Keywords
- Tctex-1, dynein light chain, RhoA activation, stress fiber formation, microtubule depolymerization, protein phosphorylation, protein-protein interaction, cytoskeleton regulation
- MeSH terms
- Animals, Dyneins, Embryo, Mammalian, Fibroblasts, Actin Cytoskeleton, Microtubules, Phosphorylation, Proto-Oncogene Proteins, Cyclic AMP-Dependent Protein Kinases, Guanine Nucleotide Exchange Factors, Mice, Rho Guanine Nucleotide Exchange Factors
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