Biology subjects

Dynlacht, B. D.

Publications and source records attributed to Dynlacht, B. D..

4 recordsLinked to original sources

bioRxiv2025

The tubulin poly-glutamylase complex, TPGC, is required for phosphatidyl inositol homeostasis and cilium assembly and maintenance

The tubulin poly-glutamylase complex (TPGC) is comprised of TTLL1 and at least five associated proteins that promote the addition of glutamate residues to tubulin tails of microtubules. Despite its discovery two decades ago, the enzyme has been refractory to characterization owing to its complex multimeric nature and the inability to detect poly-glutamylase activity after assembling the six-subunit complex. We now show that TPGC is the key enzyme driving centriolar and ciliary poly-glutamylation. We identified two novel TPGC subunits, TBC1D19 and KIAA1841, and showed that both components play an essential role in the assembly of the eight-subunit holo-enzyme. Remarkably, we were able to reconstitute the activity of TPGC with all eight subunits. TBC1D19 and KIAA1841 were essential for assembly and activity, and loss of TBC1D19 strongly compromised multiple tubulin modifications, including axonemal poly-glutamylation. TBC1D19 loss abolished transport of Arl13b and other ciliary membrane proteins, abrogating primary cilium assembly. Structural modeling revealed an essential role for TBC1D19 and KIAA1841 in complex assembly, microtubule binding, and preferential poly-glutamylation of -tubulin. We found that TBC1D19 loss abrogated the ciliary localization of phosphatidyl inositol phosphatase, INPP5E, triggering cilium instability. Ciliogenesis in TBC1D19 null cells could be restored through inhibition of a specific phosphatidyl inositol phosphate (PIP) kinase, PIP5K1c, suggesting that TBC1D19 is required to instigate and maintain PIP homeostasis during ciliogenesis. Collectively, our data show that TPGC is a multi-functional enzyme essential for cilium assembly and maintenance.

cell biology↗

bioRxiv2024

PAX fusion proteins deregulate gene networks controlling mitochondrial translation in pediatric rhabdomyosarcoma

Alveolar rhabdomyosarcoma (ARMS) patients harboring PAX3-FOXO1 and PAX7-FOXO1 fusion proteins exhibit a greater incidence of tumor relapse, metastasis, and poor survival outcome, thereby underscoring the urgent need to develop effective therapies to treat this subtype of childhood cancer. To uncover mechanisms that contribute to tumor initiation, we developed a novel muscle progenitor model and used epigenomic approaches to unravel genome re-wiring events mediated by PAX3/7 fusion proteins. Importantly, these regulatory mechanisms are conserved across established ARMS cell lines, primary tumors, and orthotopic-patient derived xenografts. Among the key targets of PAX3- and PAX7-fusion proteins, we identified a cohort of oncogenes, FGF receptors, and genes essential for mitochondrial metabolism and protein translation, which we successfully targeted in preclinical trials. Our data suggest an explanation for the relative paucity of recurring mutations in this tumor, provide a compelling list of actionable targets, and suggest promising new strategies to treat this tumor.

cancer biology↗

bioRxiv2023

A mechanism that integrates microtubule motors of opposite polarity at the kinetochore corona

Chromosome biorientation on the mitotic spindle is prerequisite to errorless genome inheritance. CENP-E (kinesin 7) and Dynein-Dynactin (DD), microtubule motors with opposite polarity, promote biorientation from the kinetochore corona, a polymeric structure whose assembly requires MPS1 kinase. The coronas building block consists of ROD, Zwilch, ZW10, and the DD adaptor Spindly (RZZS). How CENP-E and DD are scaffolded and mutually coordinated in the corona remains unclear. Here, we report near-complete depletion of RZZS and DD from kinetochores after depletion of CENP-E and the outer kinetochore protein KNL1. With inhibited MPS1, CENP-E, which we show binds directly to RZZS, is required to retain kinetochore RZZS. An RZZS phosphomimetic mutant bypasses this requirement. With active MPS1, CENP-E is dispensable for corona expansion, but strictly required for physiological kinetochore accumulation of DD. Thus, we identify the corona as an integrated scaffold where CENP-E kinesin controls DD kinetochore loading for coordinated bidirectional transport of chromosome cargo.

cell biology↗

bioRxiv2022

KIF24 controls the clustering of supernumerary centrosomes in pancreatic ductal adenocarcinoma cells

Clustering of supernumerary centrosomes, potentially leading to cell survival and chromosomal instability, is frequently observed in cancers. However, the molecular mechanisms by which centrosome clustering is controlled in cancer cells remain largely unknown. A centrosomal kinesin, KIF24, was previously shown to restrain the assembly of primary cilia in mammalian cells. Here, we revealed that KIF24 depletion suppresses multipolar spindle formation by clustering centrosomes in pancreatic ductal adenocarcinoma (PDAC) cells harboring supernumerary centrosomes. KIF24 depletion also induced hyperproliferation and improved the mitotic progression in PDAC cells. On the other hand, disruption of primary cilia failed to affect the proliferation and spindle formation in KIF24-depleted cells. These results represent a novel role of KIF24 in suppressing centrosome clustering independent of primary ciliation in centrosome-amplified PDAC cells.

cell biology↗