Biology subjects

McAlear, T. S.

Publications and source records attributed to McAlear, T. S..

4 recordsLinked to original sources

bioRxiv2023

The spindle protein CKAP2 regulates microtubule dynamics and ensures faithful chromosome segregation.

Regulation of microtubule dynamics by microtubule-associated proteins (MAPs) is essential for mitotic spindle assembly and chromosome segregation. Altered microtubule dynamics, particularly increased microtubule growth rates, were found to be a contributing factor for the development of chromosomal instability, which potentiates tumorigenesis. The MAP XMAP215/CKAP5 is the only known microtubule growth factor, and whether other MAPs regulate microtubule growth in cells is unclear. Our recent in vitro reconstitution experiments have demonstrated that Cytoskeleton-Associated Protein 2 (CKAP2) increases microtubule nucleation and growth rates, and here we find that CKAP2 is also an essential microtubule growth factor in cells. By applying CRISPR-Cas9 knock-in and knock-out as well as microtubule plus-end tracking live cell imaging, we show that CKAP2 is a mitotic spindle protein that ensures faithful chromosome segregation by regulating microtubule growth. Live cell imaging of endogenously-labelled CKAP2 showed that it localizes to the spindle during mitosis, and rapidly shifts its localization to the chromatin upon mitotic exit before being degraded. Cells lacking CKAP2 display reduced microtubule growth rates and an increased proportion of chromosome segregation errors and aneuploidy that may be a result of an accumulation of kinetochore-microtubule mis-attachments. Microtubule growth rates and chromosome segregation fidelity can be rescued upon CKAP2 expression in knock-out cells, revealing a direct link between CKAP2 expression and microtubule dynamics. Our results unveil a role of CKAP2 in regulating microtubule growth in cells and provide a mechanistic explanation for the oncogenic potential of CKAP2 misregulation. Significance statementCell division is accomplished by the assembly of a mitotic spindle composed of microtubules that segregate the chromosomes. Cells with altered microtubule dynamics frequently mis-segregate chromosomes and develop aneuploidy, which contributes to cancer development. However, how microtubule dynamics are regulated in cells is not entirely understood. Here, using CRISPR-Cas9 genome editing and live cell imaging, we find that the microtubule-associated protein CKAP2 tightly regulates microtubule growth and ensures the proper segregation of chromosomes. Cells lacking CKAP2 develop errors in chromosome segregation and aneuploidy due to a substantial decline in microtubule growth rates. The essential role of CKAP2 in the regulation of microtubule growth provides an explanation for the oncogenic potential of CKAP2 misregulation. ClassificationBiological Sciences - Cell Biology

cell biology↗

bioRxiv2023

TPPP Forms Liquid Condensates and Aggregates in Multiple System Atrophy

Oligodendrocytes have elaborate arbors of microtubules that extend toward axons and spiral around myelin sheaths. Oligodendrocytes rely on satellite organelles called Golgi outposts to nucleate new microtubules at sites far from the cell body. We now show that the Golgi outpost marker TPPP (tubulin polymerization promoting protein) forms liquid condensates that co-partition with tubulin in order to nucleate microtubules. In oligodendrocytes, TPPP forms either dynamic puncta or aberrant microtubule-associated aggregates. In Multiple System Atrophy (MSA), a sequela of histological events initiates with TPPP aggregation in myelin sheaths and terminates in perinuclear TPPP co-aggregation with alpha-synuclein (aSyn). Finally, recombinant TPPP aggregates are toxic to primary oligodendrocytes. Thus, while the liquid condensate property of TPPP facilitates microtubule nucleation, it also predisposes TPPP to aggregate in disease.

neuroscience↗

bioRxiv2021

The mitotic spindle protein CKAP2 potently increases formation and stability of microtubules

Cells increase microtubule dynamics to make large rearrangements to their microtubule cytoskeleton during cell division. Changes in microtubule dynamics are essential for the formation and function of the mitotic spindle, and misregulation can lead to aneuploidy and cancer. Using in vitro reconstitution assays we show that the mitotic spindle protein Cytoskeleton-Associated Protein 2 (CKAP2) has a strong effect on nucleation of microtubules by lowering the critical tubulin concentration 100-fold. CKAP2 increases the apparent rate constant ka of microtubule growth by 50-fold and increases microtubule growth rates. In addition, CKAP2 strongly suppresses catastrophes. Our results identify CKAP2 as the most potent microtubule growth factor to date. These finding help explain CKAP2s role as an important spindle protein, proliferation marker, and oncogene.

biophysics↗

bioRxiv2019

The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications

Microtubules are cytoskeletal structures involved in structural support, microtubule-based transport and the organization of organelles in the cells. The building blocks of the microtubule, the - and {beta}-tubulin heterodimers, polymerize into protofilaments, that associate laterally to form the hollow microtubule. There exists a specific type of microtubule structures in the cilia, termed doublet microtubules, where high stability is required for ciliary beating and function. The doublet microtubule, consisting of a complete A-tubule and a partial B-tubule maintains its stability through unique interactions at its outer and inner junctions, where the A- and B-tubules meet.\n\nUsing cryo-electron microscopy, we present the answer to the long-standing question regarding the identities, localizations and structures of the Chlamydomonas doublet microtubule inner junction proteins. Using a combination of sequence bioinformatics and mass spectrometry, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction associated protein FAP126. We show that inner junction proteins PACRG and FAP20, together with FAP52, previously unidentified FAP276, FAP106 and FAP126, form an interaction hub at the inner junction, which involves tubulin sites for post-translational modifications. We further compare the Chlamydomonas and Tetrahymena doublet microtubule structures to understand the common and species-specific features of the inner junction.

biophysics↗