Paclitaxel induces NM2 ‐dependent cellular contraction through GEF ‐ H1 dissociation from microtubules and RhoA / ROCK activation in cancer cells

Decreased cell area and increased adhesion elongation together with elevated phospho-Ser19 RLC levels are clear indicators of increased contractility.

“Decreased cell area and increased adhesion elongation together with the elevation of phospho‐Ser19 RLC levels are clear indicators of increased contractility [32, 34].”

• cites [32] Aguilar‐Cuenca R, Llorente‐González C, Chapman JR, Talayero VC, Garrido‐Casado M, Delgado‐Arévalo C, et al. Tyrosine phosphorylation of the myosin regulatory light chain controls non‐muscle myosin II assembly and function in migrating cells. Curr Biol. 2020; 30(13): 2446– 2458. 32502416 PMC7343590 sourceSupported
• cites [34] Halder G, Dupont S, Piccolo S. Transduction of mechanical and cytoskeletal cues by YAP and TAZ. Nat Rev Mol Cell Biol. 2012; 13(9): 591– 600. 22895435 sourceNot checked — no full text of the source

Inhibition of tubulin deacetylase HDAC6 using tubacin produced a significant accumulation of acetylated microtubules.

“We observed that inhibition of tubulin deacetylase HDAC6 using tubacin produced a significant accumulation of acetylated microtubules [44]”

• cites [44] Haggarty SJ, Koeller KM, Wong JC, Grozinger CM, Schreiber SL. Domain‐selective small‐molecule inhibitor of histone deacetylase 6 (HDAC6)‐mediated tubulin deacetylation. Proc Natl Acad Sci USA. 2003; 100(8): 4389– 4394. 12677000 PMC153564 sourceSupported

The kinetics of GEF-H1 dissociation from microtubules induced by PTXL were much faster than those induced by inhibitors of HDAC6.

“Importantly, the kinetics of GEF‐H1 dissociation from microtubules induced by PTXL were much faster than those induced by inhibitors of HDAC6 [21]”

• cites [21] Seetharaman S, Vianay B, Roca V, Farrugia AJ, De Pascalis C, Boeda B, et al. Microtubules tune mechanosensitive cell responses. Nat Mater. 2021; 21: 366– 377. 34663953 sourcePartly supported

  The passage indexed under “Abstract” (page unavailable) reports that Tubacin treatment released GEF-H1 from microtubules into the cytosol. The Cell treatment section (page unavailable) identifies Tubacin as a treatment, but our copy does not report PTXL-induced release or compare the kinetics of PTXL and HDAC6 inhibitors (searched: PTXL, paclitaxel, and GEF-H1 dissociation kinetics). (LLM, conf=0.85)

  Experimental — accuracy not yet measured

The C1 domain of GEF-H1 binds both intra-tubulin dimer and inter-protofilament contacts on the outer surface of the microtubule, well away from the PTXL binding site.

“Recent work from the Steinmetz group has shown that the C1 domain of GEF‐H1 binds both intra‐tubulin dimer and inter‐protofilament contacts on the outer surface of the microtubule [59], well away from the PTXL binding site [56].”

• cites [59] Choi SR, Blum TB, Giono M, Roy BK, Vakonakis I, Schmid D, et al. Structural basis of microtubule‐mediated signal transduction. Cell. 2025; 189: 461– 477. 41365297 sourceSupported
• cites [56] Prota AE, Lucena‐Agell D, Ma Y, Estevez‐Gallego J, Li S, Bargsten K, et al. Structural insight into the stabilization of microtubules by taxanes. elife. 2023; 12: e84791. 36876916 PMC10049219 sourcePartly supported

  Citation [56] is attached to the comparison with the PTXL binding site, not to the preceding account of GEF-H1 binding, which cites [59]. The Results section identifies the paclitaxel/taxane binding site on β-tubulin, but our copy does not locate GEF-H1 or establish that its binding contacts are well away from that site. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

The interaction of GEF-H1 with MTs is very sensitive to mechanical modifications to the microtubule, including changes in stiffness, curvature, or bending.

“However, structural data explicitly support that the interaction of GEF‐H1 with MTs is very sensitive to mechanical modifications to the microtubule, including changes in stiffness, curvature, or bending [59].”

• cites [59] Choi SR, Blum TB, Giono M, Roy BK, Vakonakis I, Schmid D, et al. Structural basis of microtubule‐mediated signal transduction. Cell. 2025; 189: 461– 477. 41365297 sourcePartly supported

  The Discussion describes a GEFH1 binding mode that should enable it to probe microtubule-lattice changes related to bending, stretching, twisting, and compression. That is a proposed capability, not explicit evidence that binding is very sensitive to changes in stiffness or curvature; the cited passages do not establish that degree of sensitivity. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

GEF-H1 is an active RhoGEF.

“Indeed, ARHGEF2/GEF‐H1 is an active RhoGEF [54]; thus, limiting its intrinsic activity is essential to control the contractile homeostasis of the cell.”

• cites [54] Muller PM, Rademacher J, Bagshaw RD, Wortmann C, Barth C, van Unen J, et al. Systems analysis of RhoGEF and RhoGAP regulatory proteins reveals spatially organized RAC1 signalling from integrin adhesions. Nat Cell Biol. 2020; 22(4): 498– 511. 32203420 sourceSupported

GEF‐H1 activates membrane‐bound RhoA, triggering downstream signals that elevate cellular contractility in an NM2‐dependent manner.

“GEF‐H1 then activates membrane‐bound RhoA [35], triggering downstream signals that elevate cellular contractility in an NM2‐dependent manner.”

• cites [35] Aureille J, Prabhu SS, Barnett SF, Farrugia AJ, Arnal I, Lafanechere L, et al. Focal adhesions are controlled by microtubules through local contractility regulation. EMBO J. 2024; 43(13): 2715– 2732. 38769437 PMC11217342 sourcePartly supported

  The Results section (‘Rho activation by GEF-H1 is required for focal adhesion disassembly upon microtubule targeting’; page unavailable) identifies GEF-H1’s function in this process as RhoA activation. The Discussion links that activation to a local actomyosin contractile network and traction forces. Our copy does not specify that the activated RhoA is membrane-bound, so that qualifier is unsupported. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Arp2/3‐dependent branched actin has a specific role in counterbalancing microtubule‐induced contractile tension, potentially through its coupling to nascent adhesions and the substrate.

“Instead, these results point to a specific role for Arp2/3‐dependent branched actin in counterbalancing microtubule‐induced contractile tension, potentially through its coupling to nascent adhesions and the substrate [38, 39]”

• cites [38] Alexandrova AY, Arnold K, Schaub S, Vasiliev JM, Meister JJ, Bershadsky AD, et al. Comparative dynamics of retrograde actin flow and focal adhesions: formation of nascent adhesions triggers transition from fast to slow flow. PLoS One. 2008; 3(9): e3234. 18800171 PMC2535565 sourcePartly supported

  The Discussion (page unavailable in our copy) links lamellipodial actin flow to nascent adhesions and discusses a possible Arp2/3–vinculin connection to adhesion. It does not say that branched actin counterbalances microtubule-induced contractile tension; that part of the attributed claim is unsupported by this source. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [39] Choi CK, Vicente‐Manzanares M, Zareno J, Whitmore LA, Mogilner A, Horwitz AR. Actin and alpha‐actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor‐independent manner. Nat Cell Biol. 2008; 10(9): 1039– 1050. 19160484 PMC2827253 sourceSupported

Increased contraction in vivo may lead to the acquisition of additional migratory capabilities through mesenchymal-amoeboid transitions.

“In this regard, increased contraction in vivo may lead to the acquisition of additional migratory capabilities through mesenchymal‐amoeboid transitions [70].”

• cites [70] Orgaz JL, Crosas‐Molist E, Sadok A, Perdrix‐Rosell A, Maiques O, Rodriguez‐Hernandez I, et al. Myosin II reactivation and cytoskeletal remodeling as a Hallmark and a vulnerability in melanoma therapy resistance. Cancer Cell. 2020; 37(1): 85– 103. 31935375 PMC6958528 sourcePartly supported

  The Introduction links myosin II-driven contractility to migration and aggressive amoeboid invasion, and the Discussion discusses increased myosin II activity in vivo. Neither passage says that increased contraction gives cells additional migratory capabilities through a mesenchymal-to-amoeboid transition. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

αTAT1 depletion decreases K40 α-tubulin acetylation.

“Accordingly, αTAT1 depletion decreases K40 α‐tubulin acetylation [22, 23], which should increase retention of GEF‐H1 in microtubules.”

• cites [22] Kalebic N, Sorrentino S, Perlas E, Bolasco G, Martinez C, Heppenstall PA. alphaTAT1 is the major alpha‐tubulin acetyltransferase in mice. Nat Commun. 2013; 4: 1962. 23748901 sourceSupported
• cites [23] Kim GW, Li L, Ghorbani M, You L, Yang XJ. Mice lacking alpha‐tubulin acetyltransferase 1 are viable but display alpha‐tubulin acetylation deficiency and dentate gyrus distortion. J Biol Chem. 2013; 288(28): 20334– 20350. 23720746 PMC3711300 sourceSupported

Mutation of a major α-tubulin regulatory site (K40R) markedly slowed and reduced the efficiency of PTXL-induced GEF-H1 dissociation.

“Mutation of a major α‐tubulin regulatory site (K40R) markedly slowed and reduced the efficiency of PTXL‐induced GEF‐H1 dissociation”

GEF-H1 depletion decreased NM2-B levels similarly across DMSO, VBL, and PTXL treatment conditions.

“GEF‐H1 depletion decreased NM2‐B levels similarly across DMSO, VBL, and PTXL treatment conditions”

Depletion of tubulin acetyltransferase αTAT1 induced NM2 phosphorylation.

“Unexpectedly, depletion of tubulin acetyltransferase αTAT1 also induced NM2 phosphorylation”

PTXL increased the number and size of focal adhesions in cells spread on fibronectin.

“It also increased the number and size of focal adhesions in cells spread on fibronectin (Fig. 2E–G).”

PTXL (100 nM, 3 h) promoted rapid accumulation of acetylated microtubules in U2OS osteosarcoma cells.

“These conditions promoted rapid accumulation of acetylated microtubules, as revealed by western blot using a specific antibody (Fig. 1A).”

PTXL increased RLC phosphorylation at Ser19 and Thr18/Ser19 in ovarian cancer cell lines OVCAR-8 and SK-OV-3.

“Similar results were also observed in two ovarian cancer cell lines, OVCAR‐8 and SK‐OV‐3 (Fig. 1D–G).”

VBL increased RLC phosphorylation at Ser19 in U2OS cells.

“It also increased RLC phosphorylation at Ser19 (Fig. 1B,C).”

shRNA-mediated depletion of RhoA, the downstream target of GEF-H1, impaired VBL- and PTXL-induced RLC phosphorylation.

“shRNA‐mediated depletion of RhoA, the downstream target of GEF‐H1, similarly impaired VBL‐ and PTXL‐induced RLC phosphorylation (Fig. 5A,B).”

GEF-H1 depletion abrogated the VBL- and PTXL-induced decrease in cellular area.

“GEF‐H1 depletion abrogated the VBL‐ and PTXL‐induced decrease in cellular area (Fig. 4A–C)”

GEF-H1 depletion abrogated the VBL- and PTXL-induced increase in focal adhesion number and size seen in scrambled shRNA controls.

“as well as the increase in focal adhesion number and size seen in scrambled shRNA controls (Fig. 4A,B,D–F).”

GEF-H1 silencing impaired VBL- and PTXL-induced phosphorylation at Ser19 and Thr18/Ser19.

“GEF‐H1 silencing impaired VBL‐ and PTXL‐induced phosphorylation at Ser19 and Thr18/Ser19 (Fig. 3B,C).”

PTXL (100 nM, 3 h) promoted RLC phosphorylation at Ser19 and Thr18/Ser19 in U2OS cells.

“Strikingly, PTXL also promoted RLC phosphorylation at Ser19 and Thr18/Ser19 by western blot (Fig. 1B,C).”

Co‐treatment with the ROCK inhibitor Y‐27632 and NM2 inhibitor blebbistatin negated the actin bundling effect of both PTXL and tubacin.

“Finally, co‐treatment with the ROCK inhibitor Y‐27632 and NM2 inhibitor blebbistatin negated the actin bundling effect of both PTXL and tubacin (Fig. 9A,K,L,N,O).”

AcTub‐deficient cells did not display decreased RLC phosphorylation.

“Unexpectedly, we observed that AcTub‐deficient cells did not display decreased RLC phosphorylation.”

RLC phosphorylation at Ser19 and Thr18/Ser19 by paclitaxel requires normal levels of RhoA.

“Regulatory Light Chain (RLC) phosphorylation at Ser19 and Thr18/Ser19 by paclitaxel requires normal levels of RhoA.”

After 3 h, tubacin treatment correlated with a modest, yet significant, increase in focal adhesion size.

“After 3 h, this correlated with a modest, yet significant, increase in focal adhesion size (Fig. 11C,D)”

K40R mutation in α‐tubulin partially inhibits paclitaxel‐mediated release of GEF‐H1 from microtubules.

“K40R mutation in α‐tubulin partially inhibits paclitaxel‐mediated release of GEF‐H1 from microtubules.”

Tubacin has no statistically significant effect on GEF‐H1 association with K40R microtubules.

“Tubacin had no statistically significant effect on GEF‐H1 association with K40R microtubules (Fig. 8A,C)”

Retraction and blebbing appeared more pronounced upon CK‐666/PTXL co‐treatment than CK‐666/tubacin co‐treatment.

“with retraction and blebbing appearing more pronounced upon CK‐666/PTXL co‐treatment than CK‐666/tubacin co‐treatment (Fig. 9A,E,F, blue arrows).”

LIMK phosphorylation at T508 was not induced by treatment with PTXL or tubacin.

“In addition, we found that LIMK phosphorylation at T508, used as a readout of LIMK activation [40], was not induced by treatment with PTXL or tubacin”

Microtubules remained stable throughout treatment with PTXL or tubacin in cells expressing K40R α‐tubulin.

“Microtubules remained stable throughout the treatment with PTXL or tubacin (Fig. 8A,B).”

PTXL did not promote the association of UNC45a with NM2.

“Finally, PTXL did not promote the association of UNC45a with NM2”

Tubacin failed to further increase focal adhesion maturation in HDAC6‐depleted cells.

“Consistent with a specific requirement for HDAC6 activity, tubacin failed to further increase focal adhesion maturation in HDAC6‐depleted cells (Fig. 11G–I)”

VBL‐ and PTXL‐induced dissociation of GFP‐GEF‐H1 from microtubules is almost instantaneous.

“Time‐lapse analysis using TIRF microscopy revealed that VBL‐ and PTXL‐induced dissociation of GFP‐GEF‐H1 from microtubules was almost instantaneous (Fig. 7A–D).”

GFP‐GEF‐H1 preferentially interacts with tyrosinated, dynamic microtubules over acetylated, stable microtubules.

“In addition, we observed a clear preference for GFP‐GEF‐H1 to interact with tyrosinated, dynamic microtubules over acetylated, stable microtubules (Fig. 7E,F)”

Paclitaxel decreases the co‐sedimentation of GEF‐H1 with microtubules.

“Paclitaxel decreases the co‐sedimentation of GEF‐H1 with microtubules.”

Paclitaxel does not promote the association of UNC45a to NM2‐A.

“Paclitaxel does not promote the association of UNC45a to NM2‐A.”

The effect of paclitaxel on cell size and adhesion morphology requires normal levels of GEF‐H1.

“The effect of paclitaxel on cell size and adhesion morphology requires normal levels of GEF‐H1.”

No patterning of UNC45a matching α‐tubulin or acetylated microtubules was observed.

“Conversely, we did not observe any patterning of UNC45a matching α‐tubulin or acetylated microtubules (Fig. 10C,D).”

GFP‐GEF‐H1 colocalizes with the mCherry‐decorated microtubule lattice.

“We found that GFP‐GEF‐H1 colocalized with the mCherry‐decorated microtubule lattice (Fig. 7A,B).”

The effect of Y‐27632 and blebbistatin on actin bundling was particularly prominent in the lamella, where bundled actin was largely absent.

“The effect was particularly prominent in the lamella, where bundled actin was largely absent (green arrows).”

Tubacin-induced microtubule acetylation progressively decreases GEF-H1 association.

“We also observed a progressive decrease in GEF‐H1 association when microtubules become acetylated in response to the HDAC6 inhibitor tubacin.”

Tubacin produced phosphorylation of RLC at Ser19.

“and phosphorylation of RLC at Ser19 (Fig. 1D–G)”

Tubacin induces a lagged dissociation of GEF‐H1 from microtubules compared to the near‐instantaneous effect of PTXL.

“Time‐lapse, live‐image TIRF microscopy of cells co‐expressing GFP‐GEF‐H1 and mCherry‐α‐tubulin revealed that tubacin induced a lagged dissociation of GEF‐H1 from microtubules compared to the near‐instantaneous effect of PTXL (Fig. 7A,B,D).”

Addition of VBL or PTXL to the fractionation buffer increases the extent of GEF‐H1 displacement.

“Importantly, addition of VBL or PTXL to the fractionation buffer increased the extent of the effect (Fig. 6A,B).”

HDAC6 removal modestly increased adhesion maturation.

“Consistent with published results, HDAC6 removal modestly increased adhesion maturation (Fig. 11F–I).”

αTAT1 shRNA‐transfected cells displayed significantly reduced acetylated tubulin compared to non‐transfected cells.

“Similar to cells depleted of αTAT1 using CRISPR/Cas9 gene editing [45], αTAT1 shRNA‐transfected cells displayed significantly reduced acetylated tubulin (AcTub) (Fig. 12A, light yellow and mauve arrows) compared to non‐transfected cells (Fig. 12A, red arrow).”

• cites [45] Oh S, You E, Ko P, Jeong J, Keum S, Rhee S. Genetic disruption of tubulin acetyltransferase, αTAT1, inhibits proliferation and invasion of colon cancer cells through decreases in Wnt1/β‐catenin signaling. Biochem Biophys Res Commun. 2017; 482(1): 8– 14. 27836544 sourceNot checked — no full text of the source

LIMK phosphorylation at T508 was partially inhibited by Y‐27632, a specific ROCK inhibitor.

“but was partially inhibited by Y‐27632, a specific ROCK inhibitor (Fig. 9B,C).”

PTXL induces RLC phosphorylation and NM2 activation similar to nocodazole or VBL.

“Our experiments indicate that PTXL induces RLC phosphorylation and NM2 activation similar to nocodazole or VBL, suggesting a common mechanism of downstream activation.”

Tubacin produced an increase in F‐actin filament formation.

“concomitant with an increase in F‐actin filament formation (Fig. 11A,B).”

PTXL quickly jettisons GEF-H1 from the microtubule network.

“However, our data clearly show that PTXL quickly jettisons GEF‐H1 from the microtubule network (Figs 6 and 7).”

GFP‐GEF‐H1 rapidly diffuses in the cytoplasm upon treatment with either PTXL or VBL.

“Conversely, GFP‐GEF‐H1 rapidly diffused in the cytoplasm upon treatment with either PTXL or VBL (Fig. 7A–D).”

UNC45a formed mostly linear filaments following the pattern of NM2‐A.

“examination of the subcellular localization of UNC45a, NM2‐A and microtubules in control, PTXL‐ and tubacin‐treated cells using TIRF microscopy revealed that UNC45a formed mostly linear filaments following the pattern of NM2‐A.”

PTXL induces a markedly slower and less complete dissociation of GEF‐H1 from K40R microtubules compared to wild‐type microtubules.

“PTXL induced a markedly slower and less complete dissociation of GEF‐H1 from K40R microtubules (Fig. 8A,C) compared to the rapid, near‐complete dissociation observed from wild‐type microtubules in the same experiments (Fig. 7A,B,D).”

GEF-H1 and RhoA are involved in PTXL-induced NM2 activation and cellular contraction.

“Our data also confirmed that the same mediators, GEF‐H1 and RhoA, are involved in PTXL‐induced NM2 activation and cellular contraction.”

VBL and PTXL have a similar inhibitory effect on the co‐sedimentation of GEF‐H1 with microtubules, displacing a significant portion of GEF‐H1 to the supernatant.

“We found that VBL and PTXL had a similar inhibitory effect on the co‐sedimentation of GEF‐H1 with microtubules, displacing a significant portion of GEF‐H1 to the supernatant (Fig. 6A,B).”

LIMK inhibition did not phenocopy the dramatic retraction and blebbing observed with Arp2/3 inhibition.

“Notably, despite also disrupting lamellipodial architecture, LIMK inhibition did not phenocopy the dramatic retraction and blebbing observed with Arp2/3 inhibition (Fig. 9A,H,I, orange arrows)”

The rear part of the cell still displayed actin bundles after Y‐27632 and blebbistatin treatment.

“Conversely, the rear part of the cell still displayed actin bundles”

Genetic depletion of HDAC6 with two independent shRNA sequences also increased tubulin acetylation and Ser19 RLC phosphorylation modestly.

“Genetic depletion of HDAC6 with two independent shRNA sequences also increased tubulin acetylation and Ser19 RLC phosphorylation modestly (Fig. 11E).”

AcTub‐deficient cells exhibited reduced anteroposterior polarity.

“and reduced anteroposterior polarity”

AcTub‐deficient cells exhibited prominent pSer19 RLC phosphorylation in lamellae.

“Instead, they exhibited prominent pSer19 RLC phosphorylation in lamellae”

Vehicle (DMSO)‐treated cells formed thin lamellipodia followed by a region sparsely enriched in actin bundles.

“Vehicle (DMSO)‐treated cells formed thin lamellipodia followed by a region sparsely enriched in actin bundles (Fig. 9A, a).”

Mutation of K40 to Arg partially inhibits taxane-mediated dissociation of GEF-H1.

“Interestingly, mutation of the main acetylation residue in alpha‐tubulin, K40, to Arg abrogates the releasing effect of HDAC6 inhibitors, while also partially inhibiting taxane‐mediated dissociation of GEF‐H1, suggesting that this site is important for the transmission of conformational changes involved in such release.”

Taxanes drive rapid cellular contraction.

“Our work demonstrates that taxanes drive rapid cellular contraction.”

Chronic loss of microtubule acetylation paradoxically increases rather than decreases contractility.

“Indeed, the effect of αTAT1 depletion, which reorganizes active NM2 and increases its representation in protrusive regions of the cell, illustrates that chronic loss of microtubule acetylation paradoxically increases rather than decreases contractility, pointing to a tonic homeostatic role for αTAT1 that is mechanistically distinct from the acute taxane response.”

Mutation of K40 to Arg abrogates the releasing effect of HDAC6 inhibitors.

“Interestingly, mutation of the main acetylation residue in alpha‐tubulin, K40, to Arg abrogates the releasing effect of HDAC6 inhibitors, while also partially inhibiting taxane‐mediated dissociation of GEF‐H1, suggesting that this site is important for the transmission of conformational changes involved in such release.”

K40R α-tubulin significantly impairs, but does not completely abolish, PTXL-induced GEF-H1 dissociation.

“Despite the strong experimental evidence that PTXL induces the rapid dissociation of GEF‐H1 from microtubules through a mechanism involving microtubule rigidity and/or expansion, the observation that K40R α‐tubulin significantly impairs, but does not completely abolish, PTXL‐induced GEF‐H1 dissociation warrants mechanistic consideration.”

Inhibition of HDAC6 also dissociates GEF-H1 from the microtubule lattice, albeit with much slower kinetics.

“Consistent with previous studies, we observe that inhibition of HDAC6 also dissociates GEF‐H1 from the microtubule lattice, albeit with much slower kinetics.”

Alteration of the adhesion-protrusion coupling, for example, by Arp2/3 inhibition, drives hypercontraction and blebbing.

“Alteration of the adhesion‐protrusion coupling in these conditions, for example, by Arp2/3 inhibition, drives hypercontraction and blebbing.”

Adhesions are reinforced (they grow in size) due to the increased contractility.

“As a result, adhesions are reinforced (they grow in size) due to the increased contractility.”

PTXL induced cell contraction through a mechanism involving rapid dissociation of GEF-H1 from microtubules and phosphorylation and acute activation of NM2 in a RhoA-dependent manner.

“We found that paclitaxel (PTXL) induced cell contraction through a mechanism that involved the rapid dissociation of GEF‐H1 from microtubules, and the phosphorylation and acute activation of NM2 in a RhoA‐dependent manner.”

• Also stated in: § Introduction · plain

  “similar to tubulin depolymerizers, it promotes GEF‐H1 dissociation from the microtubule lattice, increasing NM2 phosphorylation and overall contractility throughout the cell.”

Microtubule-depolymerizing agents and microtubule stabilizers, despite antagonistic effects on microtubule stability, produce comparable effects on the contractile machinery of adherent tumor cells.

“These results indicate that microtubule‐depolymerizing agents and microtubule stabilizers, despite having antagonistic effects on microtubule stability, produce comparable effects on the contractile machinery of adherent tumor cells.”

Different feedback regulatory circuits control the expression of each NM2 isoform.

“suggesting that different feedback regulatory circuits control the expression of each isoform.”

The similar decrease in NM2-B levels across conditions is consistent with an effect on NM2-B expression rather than a treatment-specific response.

“consistent with an effect on NM2‐B expression rather than a treatment‐specific response (Fig. 3E).”

K40 of α-tubulin is required for the full, rapid release of GEF-H1 from the microtubule lattice.

“indicating that this site is required for the full, rapid release of GEF‐H1 from the microtubule lattice.”

The thicker, coalescing bundles and actin arches in PTXL‐treated cells are consistent with increased NM2‐mediated contractility.

“consistent with increased NM2‐mediated contractility”

K40 acetylation is tubacin's principal mode of action in this context.

“consistent with K40 acetylation being tubacin's principal mode of action in this context.”

Reduced anteroposterior polarity is an indicator of increased isotropic contraction.

“which is an indicator of increased isotropic contraction (Fig. 12B,C).”

The homeostatic levels of tubulin acetylation mediated by αTAT1 control RLC activation and the organization of actomyosin bundles.

“These data indicate that the homeostatic levels of tubulin acetylation mediated by αTAT1 control RLC activation and the organization of actomyosin bundles.”

Microtubule‐targeting drugs increase contractility specifically through a ROCK/NM2‐dependent mechanism.

“Together, these experiments indicate that, although protrusion and contraction are interconnected processes, microtubule‐targeting drugs increase contractility specifically through a ROCK/NM2‐dependent mechanism.”

Increased activity of the existing filament pool, via the GEF‐H1/RhoA/ROCK axis, is the main operative mechanism.

“Instead, the data point to increased activity of the existing filament pool, via the GEF‐H1/RhoA/ROCK axis, as the main operative mechanism.”

Acetylation status influences the steady‐state association of GEF‐H1 with the microtubule lattice.

“consistent with acetylation status influencing the steady‐state association of GEF‐H1 with the microtubule lattice.”

Both tubacin and HDAC6 depletion converge on a shared, rate‐limiting step.

“suggesting that both perturbations converge on a shared, rate‐limiting step”

Microtubule stiffening by PTXL triggers the decrease in affinity for GEF-H1 independent of its effect on tubulin acetylation.

“Our data instead point to a mechanism by which microtubule stiffening by PTXL triggers the decrease in affinity for GEF‐H1 independent of its effect on tubulin acetylation.”

Arp2/3‐dependent dendritic actin polymerization normally counterbalances the additional contractile tension generated by microtubule‐targeting drugs, preventing hypercontraction.

“This suggests that Arp2/3‐dependent dendritic actin polymerization normally counterbalances the additional contractile tension generated by microtubule‐targeting drugs, preventing hypercontraction”

Limiting GEF-H1 intrinsic activity is essential to control the contractile homeostasis of the cell.

“Indeed, ARHGEF2/GEF‐H1 is an active RhoGEF [54]; thus, limiting its intrinsic activity is essential to control the contractile homeostasis of the cell.”

Paclitaxel and tubacin increase lamellar actomyosin bundling through ROCK/NM2 independently of LIMK activation.

“Paclitaxel and tubacin increase lamellar actomyosin bundling through ROCK/NM2 independently of LIMK activation.”

Loss of lamellipodial actin organization per se is not sufficient to unleash hypercontraction.

“suggesting that loss of lamellipodial actin organization per se is not sufficient to unleash hypercontraction.”

PTXL induces rapid cellular contraction dependent on the GEF-H1-RhoA-ROCK axis.

“Together, these results indicate that PTXL induces rapid cellular contraction dependent on the GEF‐H1‐RhoA‐ROCK axis”

The residual, slower dissociation observed on K40R microtubules reflects a K40‐independent component of the PTXL response.

“and that the residual, slower dissociation observed on K40R microtubules reflects a K40‐independent component of the PTXL response.”

LIMK/cofilin activity may primarily regulate actin filament turnover rather than substrate‐coupled force transmission.

“whereas LIMK/cofilin activity may primarily regulate actin filament turnover rather than substrate‐coupled force transmission.”

Homeostatic, αTAT1-dependent microtubule acetylation maintains appropriate levels of cellular contractility through long-term control of NM2 phosphorylation and actomyosin organization.

“whereas homeostatic, αTAT1‐dependent microtubule acetylation maintains appropriate levels of cellular contractility through long‐term control of NM2 phosphorylation and actomyosin organization.”

K40 acetylation of α‐tubulin is required for the efficient, rapid dissociation of GEF‐H1 induced by PTXL.

“Together, these data indicate that K40 acetylation of α‐tubulin is required for the efficient, rapid dissociation of GEF‐H1 induced by PTXL”

The activating effect of PTXL on NM2 is largely independent of homeostatic, αTAT1‐dependent, microtubule acetylation.

“In the context of the previous observations, this dataset further demonstrates that the activating effect of PTXL on NM2 is largely independent of homeostatic, αTAT1‐dependent, microtubule acetylation.”

PTXL acts as a stronger inducer of contractility in this system.

“This is consistent with PTXL acting as a stronger inducer of contractility in our system.”

PTXL decreases the interaction of GEF‐H1 with microtubules.

“These data indicate that PTXL also decreases the interaction of GEF‐H1 with microtubules.”

Arp2/3‐dependent branched actin polymerization counterbalances this contractile tension, likely through its mechanical coupling to adhesions.

“Arp2/3‐dependent branched actin polymerization counterbalances this contractile tension, likely through its mechanical coupling to adhesions.”

Microtubule acetylation is involved in the maintenance of contractile homeostasis.

“indicating that microtubule acetylation is involved in the maintenance of contractile homeostasis.”

Paclitaxel promotes the release of GEF‐H1 from microtubules.

“Paclitaxel promotes the release of GEF‐H1 from microtubules.”

The persistence of actin bundles in the rear part of the cell is likely due to the increased stability of these structures, which endows them with partial resistance to the inhibitors.

“likely due to the increased stability of these structures, which endows them with partial resistance to the inhibitors.”

An increase in total filament number is ruled out as the source of enhanced contractility.

“ruling out an increase in total filament number as the source of enhanced contractility.”

K40 of α-tubulin gates the efficiency of GEF-H1 dissociation from microtubules.

“in which K40 of α‐tubulin gates the efficiency of GEF‐H1 dissociation from microtubules”

The phenotype is not due to an off‐target explanation.

“and arguing against an off‐target explanation for the phenotype.”

Loss of Arp2/3‐dependent protrusive counterforce leaves contractile tension unopposed, driving the cell into a hypercontracted, blebbing state.

“loss of this protrusive counterforce (triggered by CK‐666‐induced Arp2/3 inhibition) leaves contractile tension unopposed, driving the cell into a hypercontracted, blebbing state.”

PTXL, nocodazole, and VBL share a common mechanism of downstream activation.

“Our experiments indicate that PTXL induces RLC phosphorylation and NM2 activation similar to nocodazole or VBL, suggesting a common mechanism of downstream activation.”

PTXL‐induced microtubule straightening does not translate into increased UNC45a‐dependent NM2‐A filament assembly.

“Together, these data indicate that PTXL‐induced microtubule straightening does not translate into increased UNC45a‐dependent NM2‐A filament assembly”

Binding of PTXL to the microtubule lowers its affinity for GEF-H1.

“This indicates that the binding of PTXL to the microtubule lowers its affinity for GEF‐H1.”

The electrostatic component enables rapid and complete dissociation of GEF-H1 from wild-type microtubules.

“The second component is electrostatic: acetylation‐dependent charge neutralization at K40 in the lumen facilitates the conformational propagation of the PTXL‐induced expansion to the outer surface, enabling the rapid and complete dissociation of GEF‐H1 observed from wild‐type microtubules.”

• AssumptionRests on an assumption: Acetylation of K40 neutralizes the positive charge of the lysine ε-amino group.

The K40 site is important for the transmission of conformational changes involved in GEF-H1 release.

“Interestingly, mutation of the main acetylation residue in alpha‐tubulin, K40, to Arg abrogates the releasing effect of HDAC6 inhibitors, while also partially inhibiting taxane‐mediated dissociation of GEF‐H1, suggesting that this site is important for the transmission of conformational changes involved in such release.”

αTAT1 controls microtubule dynamics independent of its tubulin acetylation activity.

“Likewise, αTAT1 controls microtubule dynamics independent of its tubulin acetylation activity, as a mutant unable to catalyze acetylation still promotes microtubule destabilization [69].”

• cites [69] Kalebic N, Martinez C, Perlas E, Hublitz P, Bilbao‐Cortes D, Fiedorczuk K, et al. Tubulin acetyltransferase alphaTAT1 destabilizes microtubules independently of its acetylation activity. Mol Cell Biol. 2013; 33(6): 1114– 1123. 23275437 PMC3592022Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

αTAT1 has a tonic homeostatic role that is mechanistically distinct from the acute taxane response.

“Indeed, the effect of αTAT1 depletion, which reorganizes active NM2 and increases its representation in protrusive regions of the cell, illustrates that chronic loss of microtubule acetylation paradoxically increases rather than decreases contractility, pointing to a tonic homeostatic role for αTAT1 that is mechanistically distinct from the acute taxane response.”

K40R mutant permanently mimics the unacetylated state of K40 regardless of αTAT1 activity.

“Importantly, while arginine also bears a positive charge at physiological pH, it cannot be acetylated, and therefore permanently mimics the unacetylated state of K40 regardless of αTAT1 activity.”

• AssumptionRests on an assumption: Arginine cannot be acetylated and therefore permanently mimics the unacetylated state of K40 regardless of αTAT1 activity.

HDAC6 and αTAT1 may each control contractility, at least in part, through a tubulin acetylation-independent mechanism.

“Our data thus suggest that the effect of αTAT1 depletion on actomyosin contractility may go beyond its role in tubulin acetylation/deacetylation, and that HDAC6 and αTAT1 may each control contractility, at least in part, through a tubulin acetylation‐independent mechanism.”

GEF-H1 compartmentalization is part of a homeostatic mechanism, acting in conjunction with protrusion-adhesion at the lamellipodium, that prevents hypercontraction.

“Our experiments also revealed that GEF‐H1 compartmentalization is part of a homeostatic mechanism, acting in conjunction with protrusion‐adhesion at the lamellipodium, that prevents hypercontraction.”

The effect of αTAT1 depletion on actomyosin contractility may go beyond its role in tubulin acetylation/deacetylation.

“Our data thus suggest that the effect of αTAT1 depletion on actomyosin contractility may go beyond its role in tubulin acetylation/deacetylation, and that HDAC6 and αTAT1 may each control contractility, at least in part, through a tubulin acetylation‐independent mechanism.”

The requirement for both components may represent a regulatory safeguard, ensuring that GEF-H1 is not released accidentally by either mechanical or chemical perturbation of the microtubule lattice alone, thereby preventing an inappropriate activation of the RhoA/ROCK/NM2 axis.

“The requirement for both components may represent a regulatory safeguard, ensuring that GEF‐H1 is not released accidentally by either mechanical or chemical perturbation of the microtubule lattice alone, thereby preventing an inappropriate activation of the RhoA/ROCK/NM2 axis.”

Taxanes have a novel effect that may be involved in the development of resistance to therapy and potential invasiveness by altering the contractile homeostasis of resistant cells.

“Overall, this work highlights a novel effect of taxanes that may be involved in the development of resistance to therapy and potential invasiveness by altering the contractile homeostasis of resistant cells.”

The electrostatic consequence of acetylation (charge neutralization) is a key determinant of GEF-H1 dissociation efficiency.

“This distinction argues against a simple steric interpretation of the K40R phenotype and instead points to the electrostatic consequence of acetylation (that is, charge neutralization) as a key determinant of GEF‐H1 dissociation efficiency.”

• AssumptionRests on an assumption: Acetylation of K40 neutralizes the positive charge of the lysine ε-amino group.

HDAC6 has catalytic-dependent and -independent functions.

“In addition, we showed that HDAC6 has catalytic‐dependent and ‐independent functions [68].”

• cites [68] Cabrero JR, Serrador JM, Barreiro O, Mittelbrunn M, Naranjo‐Suarez S, Martin‐Cofreces N, et al. Lymphocyte chemotaxis is regulated by histone deacetylase 6, independently of its deacetylase activity. Mol Biol Cell. 2006; 17(8): 3435– 3445. 16738306 PMC1525231 sourceNot checked — no full text of the source

Tubulin is the major substrate of HDAC6 involved in the GEF-H1 dissociation effect.

“although the lack of effect of tubacin on GEF‐H1 dissociation from K40R microtubules strongly argues that tubulin is the major substrate of HDAC6 involved in this effect.”

Decreased K40 acetylation should increase retention of GEF-H1 in microtubules.

“Accordingly, αTAT1 depletion decreases K40 α‐tubulin acetylation [22, 23], which should increase retention of GEF‐H1 in microtubules.”

The mechanical component alone is sufficient for partial GEF-H1 release, as evidenced by residual dissociation from K40R microtubules.

“This mechanical component alone is sufficient for partial GEF‐H1 release, as evidenced by the residual dissociation observed from K40R microtubules.”