Bachelor of Science, Nanjing University (2012)
Doctor of Philosophy, University of Massachusetts Amherst (2018)
Alarice Lowe, Postdoctoral Faculty Sponsor
In this protocol, we describe a simple microscopy-based method to assess the interaction of a microtubule-associated protein (MAP) with microtubules. The interaction between MAP and microtubules is typically assessed by a co-sedimentation assay, which measures the amount of MAP that co-pellets with microtubules by centrifugation, followed by SDS-PAGE analysis of the supernatant and pellet fractions. However, MAPs that form large oligomers tend to pellet on their own during the centrifugation step, making it difficult to assess co-sedimentation. Here we describe a microscopy-based assay that measures microtubule binding by direct visualization using fluorescently-labeled MAP, solving the limitations of the co-sedimentation assay. Additionally, we recently reported quantification of microtubule bundling by measuring the thickness of individual microtubule structures observed in the microscopy-based assay, making the protocol more advantageous than the traditional microtubule co-pelleting assay.
View details for DOI 10.21769/BioProtoc.3110
View details for Web of Science ID 000458028500014
View details for PubMedID 30733975
View details for PubMedCentralID PMC6363367
Dynein mediates spindle positioning in budding yeast by pulling on astral microtubules (MTs) from the cell cortex. The MT-associated protein She1 regulates dynein activity along astral MTs and directs spindle movements toward the bud cell. In addition to localizing to astral MTs, She1 also targets to the spindle, but its role on the spindle remains unknown. Using function-separating alleles, live-cell spindle assays, and in vitro biochemical analyses, we show that She1 is required for the maintenance of metaphase spindle stability. She1 binds and cross-links MTs via a C-terminal MT-binding site. She1 can also self-assemble into ring-shaped oligomers. In cells, She1 stabilizes interpolar MTs, preventing spindle deformations during movement, and we show that this activity is regulated by Ipl1/Aurora B phosphorylation during cell cycle progression. Our data reveal how She1 ensures spindle integrity during spindle movement across the bud neck and suggest a potential link between regulation of spindle integrity and dynein pathway activity.
View details for DOI 10.1083/jcb.201701094
View details for Web of Science ID 000409075500019
View details for PubMedID 28794129
View details for PubMedCentralID PMC5584168
Aspergillus nidulans is an ideal model to study nuclear migration and intracellular transport by dynein and kinesin owing to its long neuron-like hyphae, conserved transport mechanisms, and powerful genetics. In this organism, as in other filamentous fungi, microtubules have been implicated in patterning cell shape through polarized tip growth - the hallmark mode of growth that generates the elongated hyphae. Exactly how microtubules regulate tip growth is incompletely understood and remains a fascinating question for various cell types, such as pollen tubes and root hairs. Zeng et?al. (2014) describe important new findings in A. nidulans regarding the role of EBA, the master regulator of microtubule plus end-tracking proteins, in specifying microtubule dynamics required for directional tip growth at the hyphal tip.
View details for DOI 10.1111/mmi.12791
View details for Web of Science ID 000344465000002
View details for PubMedID 25213368
View details for PubMedCentralID PMC4213288