Biology subjects

Thompson, A. F.

Publications and source records attributed to Thompson, A. F..

4 recordsLinked to original sources

bioRxiv2026

A Phosphorylation Switch Governs KIF11's Mechanical Output During Mitosis

The kinesin-5 motor protein KIF11 is crucial for mitotic spindle assembly, driving the separation of spindle poles through microtubule sliding. Src-family kinases phosphorylate KIF11 at multiple tyrosine residues within its motor domain, but the mechanistic consequences of these modifications remain unclear. Here, we dissect the role of phosphorylation at Y211 using phospho-mimetic (Y211E) and non-phosphorylatable mutants (Y211F) in biochemical, biophysical, and cellular assays. Optical trapping and Forster resonance energy transfer (FRET) analyses reveal that Y211 phosphorylation slows neck-linker docking, reducing motor velocity and force generation under load. In human cells, Y211E expression impairs bipolar spindle formation and decreases spindle pole separation velocity, while Y211F shortens steady-state spindle length. Fluorescence recovery after photobleaching (FRAP) analyses show that Y211E accelerates motor turnover on spindle microtubules, consistent with the mutant motors heightened load sensitivity. Together, these findings support a model in which Src-mediated phosphorylation at Y211 acts as a rheostat to tune KIF11 mechanochemistry and spindle assembly dynamics, linking cancer-relevant kinase signaling to mitotic force generation.

cell biology↗

bioRxiv2022

Reconstitution of renal cyst formation in 3D culture reveals a role for AMOT and Yap1 in determining cyst size.

Despite substantial progress in identifying and understanding causative mutations in autosomal dominant polycystic kidney disease (ADPKD), little is known about subsequent cellular events leading to cyst formation. In prior studies we reported that Cadherin 8, a type II Cadherin, expression is sufficient to induce cyst emergence from HK-2 cells grown as tubule arrays in collagen matrix (1). However, emergent cysts did not exhibit the luminal enlargement observed in ADPKD. In this communication, we reconstitute cyst emergence with consequent cyst lumen expansion in 3D culture by stable co-expression of Cadherin 8 in combination with a constitutively active mutant of YAP1, the key effector of the HIPPO pathway. Specifically, immortalized cells derived from ADPKD cyst epithelia formed cysts with substantially larger lumen sizes when transduced with YAP1-5SA. Conversely, expression of the YAP1 inhibitor, AMOTL1, in these cells resulted in their forming cysts with smaller lumens than control cells. Our data show that cyst formation results from a sequential two-step process consisting of cyst initiation and subsequent cyst expansion. Taken together, cyst initiation induced by Cadherin 8 expression is proposed to result from decreased cell-cell adhesion while cyst expansion is driven by increased Yap1 activity.

cell biology↗

bioRxiv2021

Pathogenic mutations in the chromokinesin KIF22 disrupt anaphase chromosome segregation

The chromokinesin KIF22 generates forces that contribute to mitotic chromosome congression and alignment. Mutations in the 2 helix of the motor domain of KIF22 have been identified in patients with abnormal skeletal development, and we report the identification of a patient with a novel mutation in the KIF22 tail. We demonstrate that pathogenic mutations do not result in a loss of KIF22s functions in early mitosis. Instead, mutations disrupt chromosome segregation in anaphase, resulting in reduced proliferation, abnormal daughter cell nuclear morphology, and, in a subset of cells, cytokinesis failure. This phenotype could be explained by a failure of KIF22 to inactivate in anaphase. Consistent with this model, constitutive activation of the motor via a known site of phosphoregulation in the tail phenocopied the effects of pathogenic mutations. These results suggest the motor domain 2 helix may be an important site for regulation of KIF22 activity at the metaphase to anaphase transition. In support of this conclusion, mimicking phosphorylation of 2 helix residue T158 also prevents inactivation of KIF22 in anaphase. These findings demonstrate the importance of both the head and tail of the motor in regulating the activity of KIF22 and offer insight into the cellular consequences of preventing KIF22 inactivation and disrupting force balance in anaphase.

cell biology↗

bioRxiv2019

The Kinesin-5 Tail Domain Directly Modulates the Mechanochemical Cycle of the Motor for Anti-Parallel Microtubule Sliding

Kinesin-5 motors organize mitotic spindles by sliding apart anti-parallel microtubules. They are homotetramers composed of two antiparallel dimers placing orthogonal motor and tail domains at opposite ends of a bipolar minifilament. Here, we describe a regulatory mechanism, involving direct binding of the tail to motor domain and reveal its fundamental role in microtubule sliding motility. Biochemical analyses reveal that the tail down-regulates microtubule-activated ATP hydrolysis by specifically engaging the motor in the nucleotide-free or ADP-bound states. Cryo-EM structures reveal that the tail stabilizes a unique conformation of the motor N-terminal subdomain opening its active site. Full-length kinesin-5 motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering along microtubules. The tail is critical for motors to zipper together two microtubules by generating substantial forces within sliding zones. The tail domain is essential for kinesin-5 mitotic spindle localization in vivo, which becomes severely reduced when the tail is deleted. Our studies suggest a revised microtubule-sliding model, in which tail domains directly engage motor domains at both ends of kinesin-5 homotetramers enhancing stability of the dual microtubule-bound states leading to slow motility yet high force production.

biophysics↗