Biology subjects

Stumpff, J.

Publications and source records attributed to Stumpff, J..

17 recordsLinked to original sources

bioRxiv2026

A systems-level proteomic analysis identifies kinesin targets of KIFBP during neuronal development

Kinesins are molecular motor proteins essential for organizing and remodeling the cytoskeleton during neuronal development and maintenance. One key regulator is kinesin family binding protein (KIFBP), which inhibits a subset of kinesins by blocking motor-microtubule interactions. Homozygous mutations in KIFBP cause Goldberg-Shprintzen Syndrome (GOSHS), a neurodevelopmental disorder characterized by intellectual disability, microcephaly, and axonal neuropathy. Although loss of KIFBP has been linked to reduced neurite length and microtubule disorganization, the specific kinesins underlying these phenotypes remain unclear. Here we use a CRISPR-Cas9 generated KIFBP knockout Neuro-2a cell line to demonstrate that KIFBP is required for neurite extension and use inducible GFP-KIFBP to define the KIFBP interactome during neuronal differentiation. Immunoprecipitation coupled with mass spectrometry identified both known and novel KIFBP-associated kinesins. Single molecule TIRF microscopy confirmed direct inhibition of a subset of kinesins that co-immunoprecipitated with KIFBP. Notably, we identified KIF5A and KIF18B as previously unrecognized regulatory targets with potential roles in neuronal development. Together, these findings establish Neuro-2a cells as a model for studying KIFBP function and provide new insight into the regulation of kinesin activity and cytoskeletal dynamics in neurons.

cell biology↗

bioRxiv2026

KIF18A Maintains Kinetochore-Microtubule Attachments in CIN Cells by Limiting Microtubule Polymerization

Chromosomal instability (CIN) generates vulnerabilities that can be therapeutically exploited, including sensitivity to inhibition of the kinesin motor KIF18A. However, the mechanistic basis for why a subset of CIN tumor cells depend on KIF18A remains unclear. Here, we compare mitotic phenotypes across KIF18A-sensitive and -insensitive cell models. In sensitive CIN cells, KIF18A inhibition leads to formation of polar chromosomes with unattached kinetochores, recruitment of spindle assembly checkpoint proteins, and prolonged mitotic arrest. Although KIF18A loss reduces kinetochore-microtubule stability in all cell lines, sensitive cells exhibit lower baseline attachment stability and heightened microtubule polymerization rates, predisposing them to attachment failure. Acute KIF18A inhibition disrupts maintenance of attachments after metaphase alignment, while reducing microtubule polymerization suppresses mitotic defects. These findings support a model in which CIN tumor cells rely on KIF18A to restrain excessive microtubule dynamics and maintain attachment.

cancer biology↗

bioRxiv2026

Novel KIF22 Variants Disrupt Mitosis in Human Chondrocytes and Expand SEMDJL2 Mechanisms

Spondyloepimetaphyseal dysplasia with joint laxity, type 2 (SEMDJL2) is a rare skeletal disorder caused by pathogenic variants in KIF22, a mitotic chromokinesin that generates polar ejection forces (PEF) to ensure proper chromosome alignment and segregation. Although prior work showed that SEMDJL2-associated KIF22 hotspot variants impair chromosome segregation in epithelial cells, how these variants affect chondrocyte mitosis remains incompletely understood. Here, we analyzed the effects of the hotspot variant R149Q, a recently reported recessive variant R49Q, and two newly identified heterozygous variants, P144T and E222Q, in human chondrocytes. Both novel variants were identified in individuals with classic SEMDJL2 features. P144T and E222Q retained PEF-generating activity, whereas R49Q displayed reduced PEFs, consistent with their respective inheritance patterns. Live cell imaging revealed that all variants disrupted mitosis. The heterozygous variants (P144T, E222Q, R149Q) dominantly impeded anaphase chromosome segregation and spindle pole separation, supporting reclassification of P144T and E222Q as likely pathogenic. In contrast, R49Q caused milder, partially penetrant segregation defects, consistent with reduced and dysregulated motor activity. Together, our results define two mechanistic classes of KIF22 dysregulation: constitutive activation in heterozygous variants, which fail to down-regulate KIF22 at anaphase onset, and mixed-state dysregulation in the recessive R49Q variant, which exhibits partial loss of polar ejection force activity coupled with incomplete inactivation during anaphase. These findings broaden the mechanistic framework for how KIF22 variants perturb mitosis in chondrocytes and expand the genotypic landscape associated with SEMDJL2.

cell biology↗

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↗

bioRxiv2024

Anapc5 and Anapc7 as genetic modifiers of KIF18A function in fertility and mitotic progression

The kinesin family member 18A (KIF18A) is an essential regulator of microtubule dynamics and chromosome alignment during mitosis. Functional dependency on KIF18A varies by cell type and genetic context but the heritable factors that influence this dependency remain unknown. To address this, we took advantage of the variable penetrance observed in different mouse strain backgrounds to screen for loci that modulate germ cell depletion in the absence of KIF18A. We found a significant association at a Chr5 locus where anaphase promoting complex subunits 5 (Anapc5) and 7 (Anapc7) were the top candidate genes. We found that both genes were differentially expressed in a sensitive strain background when compared to resistant strain background at key timepoints in gonadal development. We also identified a novel retroviral insertion in Anapc7 that may in part explain the observed expression differences. In cell line models, we found that depletion of KIF18A induced mitotic arrest, which was partially rescued by co-depletion of ANAPC7 (APC7) and exacerbated by co-depletion of ANAPC5 (APC5). These findings suggest that differential expression and activity of Anapc5 and Anapc7 may influence sensitivity to KIF18A depletion in germ cells and CIN cells, with potential implications for optimizing antineoplastic therapies.

cell biology↗

bioRxiv2023

Identification of the KIF18A alpha-4 helix as a therapeutic target for chromosomally unstable tumor cells

The mitotic kinesin, KIF18A, is required for proliferation of cancer cells that exhibit chromosome instability (CIN), implicating it as a promising target for treatment of a subset of aggressive tumor types. Determining regions of the KIF18A protein to target for inhibition will be important for the design and optimization of effective small molecule inhibitors. In this study, we investigated the effects of mutating S284 within the alpha-4 helix of KIF18A, which was previously identified as a phosphorylated residue. Mutations in S284 cause relocalization of KIF18A from the plus-ends of spindle microtubules to the spindle poles. Furthermore, KIF18A S284 mutants display loss of KIF18A function and fail to support proliferation in CIN tumor cells. Interestingly, similar effects on KIF18A localization and function were seen after treatment of CIN cells with KIF18A inhibitory compounds that are predicted to interact with residues within the alpha-4 helix. These data implicate the KIF18A alpha-4 helix as an effective target for inhibition and demonstrate that small molecules targeting KIF18A selectively limit CIN tumor cell proliferation and result in phenotypically similar effects on mitosis at the single cell level compared to genetic perturbations.

cancer biology↗

bioRxiv2023

Modification of the Neck Linker of KIF18A Alters Microtubule Subpopulation Preference

Kinesins support many diverse cellular processes, including facilitating cell division through mechanical regulation of the mitotic spindle. However, how kinesin activity is controlled to facilitate this process is not well understood. Interestingly, post-translational modifications have been identified within the enzymatic region of all 45 mammalian kinesins, but the significance of these modifications has gone largely unexplored. Given the critical role of the enzymatic region in facilitating nucleotide and microtubule binding, it may serve as a primary site for kinesin regulation. Consistent with this idea, a phosphomimetic mutation at S357 in the neck- linker of KIF18A alters the localization of KIF18A within the spindle from kinetochore microtubules to peripheral microtubules. Changes in localization of KIF18A-S357D are accompanied by defects in mitotic spindle positioning and the ability to promote mitotic progression. This altered localization pattern is mimicked by a shortened neck-linker mutant, suggesting that KIF18A-S357D may cause the motor to adopt a shortened neck-linker like state that prevents KIF18A from accumulating at the plus-ends of kinetochore microtubules. These findings demonstrate that post-translational modifications in the enzymatic region of kinesins could be important for biasing their localization to particular microtubule subpopulations.

cell biology↗

bioRxiv2022

The histone H3.3 K27M mutation found in diffuse midline gliomas coordinately disrupts adjacent H3.3 Ser31 phosphorylation and the fidelity of chromosome segregation

During the cell cycle, differential phosphorylation of select histone H3 serine/threonine residues regulates chromatin structure, necessary for both dynamic transcriptional control and proper chromosome segregation1-2. Histone H3.3 contains a highly conserved serine residue (Ser31) within its N-terminal tail that is unique to this variant. During interphase phosphorylation of Ser31 amplifies stimulation-induced transcription and is required for early metazoan development3-6. During mitosis Ser31 phosphorylation at the pericentromere supports proper chromosome segregation, albeit by unknown mechanisms7-10. H3.3 Ser31 is flanked by mutational sites that drive several human cancers, including pediatric gliomas5-8. This is typified by the H3.3K27M mutation found in [~]80% of diffuse midline gliomas, which undergo epigenetic reprogramming in proliferative cells coordinate with loss of global H3 lysine 27 trimethylation (H3K27Me3)11-14. However, whether the K27M mutation influences the neighboring Ser31 phosphorylation and whether disrupting Ser31 phosphorylation plays a distinct role in driving gliomagenesis has not been tested. Here we show that H3.3K27M mutant cells have reduced capacity for H3.3 Ser31 phosphorylation at the mitotic pericentromere, increased rates of chromosome missegregation, and impaired G1 checkpoint responses to chromosome instability. CRISPR-reversion of K27M to wild-type restores phospho-Ser31 levels and suppresses chromosome segregation defects. CRISPR editing to introduce a non-phosphorylatable H3.3S31A alone is sufficient to increase the frequency of chromosome missegregations. Finally, expression of H3.3S31A in a PDGF{beta}-driven RCAS/TVA mouse model is sufficient to drive high grade gliomagenesis, generating diffuse tumors morphologically indistinguishable from those generated by H3.3K27M expression. Importantly, this occurs without the loss of H3K27 triple methylation that is the hallmark of K27M tumors. Our results reveal that the H3.3 K27M mutation alters the neighboring Ser31 phosphorylation, and loss of proper H3.3 Ser31 phosphorylation contributes to the formation of diffuse midline gliomas.

cancer 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↗

bioRxiv2021

Kinesin-binding protein remodels the kinesin motor to prevent microtubule-binding

Kinesins are tightly regulated in space and time to control their activation in the absence of cargo-binding. Kinesin-binding protein (KIFBP) was recently discovered to bind the catalytic motor heads of 8 of the 45 known kinesin superfamily members and inhibit binding to microtubules. In humans, mutation of KIFBP gives rise to Goldberg-Shprintzen syndrome (GOSHS), but the kinesin(s) that is misregulated to produce clinical features of the disease is not known. Understanding the structural mechanism by which KIFBP selects its kinesin binding partners will be key to unlocking this knowledge. Using a combination of cryo-electron microscopy and crosslinking mass spectrometry, we determined structures of KIFBP alone and in complex with two mitotic kinesins, revealing regions of KIFBP that participate in complex formation. KIFBP adopts an alpha-helical solenoid structure composed of TPR repeats. We find that KIFBP uses a 2-pronged mechanism to remodel kinesin motors and block microtubule-binding. First, KIFBP engages the microtubule-binding interface and sterically blocks interaction with microtubules. Second, KIFBP induces allosteric conformational changes to the kinesin motor head that displace a key structural element in the kinesin motor head (-helix 4) required for microtubule binding. We identified two regions of KIFBP necessary for in vitro kinesin-binding as well as cellular regulation during mitosis. Taken together, this work establishes the mechanism of kinesin inhibition by KIFBP and provides the first example of motor domain remodeling as a means to abrogate kinesin activity.

cell biology↗

bioRxiv2020

Micronuclei arising due to loss of KIF18A form stable micronuclear envelopes and do not promote tumorigenesis

Micronuclei, whole or fragmented chromosomes which are spatially separated from the main nucleus, are strongly associated with genomic instability and have been identified as drivers of tumorigenesis. Paradoxically, Kif18a mutant mice produce micronuclei due to unaligned chromosomes in vivo but do not develop spontaneous tumors, raising questions about whether all micronuclei contribute similarly to genomic instability and cancer. We report here that micronuclei in Kif18a mutant mice form stable nuclear envelopes. Challenging Kif18a mutant mice via deletion of the Trp53 gene led to formation of thymic lymphoma with elevated levels of micronuclei. However, loss of Kif18a had modest or no effect on survival of Trp53 homozygotes and heterozygotes, respectively. To further explore micronuclear envelope stability in KIF18A KO cells, we compared micronuclei induced via different insults in cultured cells. Micronuclei in KIF18A KO cells form stable nuclear envelopes characterized by increased recruitment of core and non-core nuclear envelope components and successful expansion of decondensing chromatin compared to those induced by microtubule drug washout or exposure to radiation. We also observed that lagging chromosomes, which lead to micronucleus formation, were positioned closer to the main chromatin masses, and further from the central spindle, in KIF18A KO cells. Our studies provide in vivo support to models suggesting that micronuclear fate depends on the sub-cellular location of late lagging chromosomes and suggest that not all micronuclei actively promote tumorigenesis.

cell biology↗

bioRxiv2020

Selective vulnerability of aneuploid human cancer cells to inhibition of the spindle assembly checkpoint

Selective targeting of aneuploid cells is an attractive strategy for cancer treatment. Here, we mapped the aneuploidy landscapes of ~1,000 human cancer cell lines and classified them by their degree of aneuploidy. Next, we performed a comprehensive analysis of large-scale genetic and chemical perturbation screens, in order to compare the cellular vulnerabilities between near-diploid and highly-aneuploid cancer cells. We identified and validated an increased sensitivity of aneuploid cancer cells to genetic perturbation of core components of the spindle assembly checkpoint (SAC), which ensures the proper segregation of chromosomes during mitosis. Surprisingly, we also found highly-aneuploid cancer cells to be less sensitive to short-term exposures to multiple inhibitors of the SAC regulator TTK. To resolve this paradox and to uncover its mechanistic basis, we established isogenic systems of near-diploid cells and their aneuploid derivatives. Using both genetic and chemical inhibition of BUB1B, MAD2 and TTK, we found that the cellular response to SAC inhibition depended on the duration of the assay, as aneuploid cancer cells became increasingly more sensitive to SAC inhibition over time. The increased ability of aneuploid cells to slip from mitotic arrest and to keep dividing in the presence of SAC inhibition was coupled to aberrant spindle geometry and dynamics. This resulted in a higher prevalence of mitotic defects, such as multipolar spindles, micronuclei formation and failed cytokinesis. Therefore, although aneuploid cancer cells can overcome SAC inhibition more readily than diploid cells, the proliferation of the resultant aberrant cells is jeopardized. At the molecular level, analysis of spindle proteins identified a specific mitotic kinesin, KIF18A, whose levels were drastically reduced in aneuploid cancer cells. Aneuploid cancer cells were particularly vulnerable to KIF18A depletion, and KIF18A overexpression restored the sensitivity of aneuploid cancer cells to SAC inhibition. In summary, we identified an increased vulnerability of aneuploid cancer cells to SAC inhibition and explored its cellular and molecular underpinnings. Our results reveal a novel synthetic lethal interaction between aneuploidy and the SAC, which may have direct therapeutic relevance for the clinical application of SAC inhibitors.

cancer biology↗

bioRxiv2020

Chromosomally unstable tumor cells specifically require KIF18A for proliferation

Chromosomal instability (CIN), characterized by frequent missegregation of chromosomes during mitosis, is a hallmark of tumor cells caused by changes in the dynamics and control of microtubules that comprise the mitotic spindle1-3. Thus, CIN tumor cells may respond differently than normal diploid cells to treatments that target mitotic spindle regulation. We tested this idea by inhibiting a subset of kinesin motor proteins that control spindle microtubule dynamics and mechanics but are not required for the proliferation of near-diploid cells. Our results indicated that KIF18A was required for proliferation of CIN cells derived from triple negative breast cancer or colorectal cancer tumors but was not required in near-diploid cells. CIN tumor cells exhibited mitotic delays, multipolar spindles due to centrosome fragmentation, and increased cell death following inhibition of KIF18A. Sensitivity to KIF18A knockdown was strongly correlated with centrosome fragmentation, which required dynamic microtubules but did not depend on bipolar spindle formation or mitotic arrest. Our results indicate the altered spindle microtubule dynamics characteristic of CIN tumor cells can be exploited to reduce the proliferative capacity of CIN cells.

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↗

bioRxiv2018

The Human Kinesin Kif18A’s Neck Linker Permits Navigation of Microtubule Bound Obstacles within the Mitotic Spindle

Mitotic chromosome alignment is essential for the robust separation of genetic material into daughter cells. In mammalian cells, this process requires the function of Kif18A, a kinesin-8 motor protein. Kif18A confines chromosome movement to the mitotic spindle equator by accumulating at the plus-ends of kinetochore microtubule bundles (K-fibers), where it functions to suppress K-fiber dynamics. It is not understood how the motor accumulates at K-fiber plus-ends, a difficult feat requiring the motor to navigate protein dense microtubule tracks. Our data indicate that Kif18As relatively long (17 amino acid) neck linker is required for the motors accumulation at K-fiber plus-ends. Shorter neck linker (sNL) variants of Kif18A display a deficiency in K-fiber accumulation, especially on K-fibers near the center of the spindle. This pattern correlates with the more uniform concentration of the microtubule bundling protein HURP on central K-fibers compared to peripheral K-fibers. Depletion of HURP permits Kif18A sNL to accumulate on central K-fibers, while HURP overexpression reduces wild-type Kif18As ability to accumulate on this same K-fiber subset. Furthermore, single molecule assays indicate that Kif18A sNL motors are less proficient at navigating microtubules coated with the microtubule associated protein tau. Taken together, these results support a model in which Kif18As neck linker length permits efficient navigation of obstacles such as HURP to reach K-fiber ends during mitosis.\n\nSignficiance StatementKinesin motor proteins play key roles in controlling chromosome alignment and segregation during cell division. The kinesin Kif18A confines chromosomes to the middle of the spindle by accumulating at the ends of microtubules attached to chromosomes. We show here that Kif18As ability to accumulate at the end of these microtubules requires navigation of microtubule-associated protein obstacles, and that this activity is imparted by a relatively long neck linker region. These findings demonstrate a molecular mechanism for navigation of densely populated microtubules inside a cell.

cell biology↗

bioRxiv2018

Mitotic chromosome alignment is required for proper nuclear envelope reassembly

Chromosome alignment at the equator of the mitotic spindle is a highly conserved step during cell division, however, its importance to genomic stability and cellular fitness are not understood. Normal mammalian somatic cells lacking Kif18A function complete cell division without aligning chromosomes. These alignment-deficient cells display normal chromosome copy numbers in vitro and in vivo, suggesting that chromosome alignment is largely dispensable for maintenance of euploidy. However, we find that loss of chromosome alignment leads to interchromosomal compaction defects during anaphase, abnormal organization of chromosomes into a single nucleus at mitotic exit, and the formation of micronuclei in vitro and in vivo. These defects slow cell proliferation and reduce postnatal growth and survival with variable penetrance in mice. Our studies support a model in which the alignment of mitotic chromosomes promotes proper nuclear envelope reassembly and continued proliferation by ensuring that chromosomes segregate as a compact mass during anaphase.

cell biology↗

bioRxiv2018

Kinesin Binding Protein (KBP) buffers the activity of Kif18A and Kif15 in mitosis to ensure accurate chromosome segregation

Mitotic kinesins must be regulated to ensure a precise balance of spindle forces and accurate segregation of chromosomes into daughter cells. Here we demonstrate that Kinesin-Binding Protein (KBP) reduces the activity of KIF18A and KIF15 during metaphase. Overexpression of KBP disrupts the movement and alignment of mitotic chromosomes and decreases spindle length, a combination of phenotypes observed in cells deficient for KIF18A and KIF15, respectively. We show through gliding filament and microtubule co-pelleting assays that KBP directly inhibits KIF18A and KIF15 motor activity by preventing microtubule-binding. Consistent with these effects, the mitotic localizations of KIF18A and KIF15 are altered by overexpression of KBP. Cells depleted of KBP exhibit lagging chromosomes in anaphase, an effect that is recapitulated by KIF15 and KIF18A overexpression. Based on these data, we propose a model in which KBP acts as a protein buffer in mitosis, protecting cells from excessive KIF18A and KIF15 activity to promote accurate chromosome segregation.\n\nSUMMARYKinesin-Binding Protein (KBP) is identified as a regulator of the kinesins KIF18A and KIF15 during mitosis. KBP buffers the activity of these motors to control chromosome alignment and spindle integrity in metaphase and prevent lagging chromosomes in anaphase.

cell biology↗