Proximity labeling at non-centrosomal microtubule-organizing centers in C. elegans epithelial cells reveals mechanisms of microtubule regulation
Abstract/Contents
- Abstract
- Microtubules are polarized polymers with dynamic plus ends and relatively stable minus ends and are critical for many cell functions. Microtubule organizing centers (MTOCs) are sites that control the growth and specific spatial assembly of microtubules from their minus ends. Research on MTOCs has mostly focused on the centrosome, an organelle that acts as an MTOC in dividing animal cells. As cells differentiate, centrosomes are often inactivated as a functioning MTOC and MTOC activity is then assigned to non-centrosomal sites (ncMTOCs). Despite the ubiquity of ncMTOCs across most tissues, knowledge of their composition, critical regulators, and mechanisms of assembly remain scarce. As a model to study MTOCs, we use the C. elegans embryonic intestinal epithelial cells, as their MTOCs can be tracked in real time and in their native environment. Once intestinal cells are polarized, the centrosome is completely inactivated, and microtubules are patterned in parallel arrays with their minus ends oriented from the 'apical ncMTOC'. Interestingly, depleting all known microtubule minus end proteins (ɣ-TuRC, PTRN 1/CAMSAP, and NOCA-1/Ninein) did not affect the density of non-centrosomal microtubules in intestinal cells. We thus used the intestine as a test bed for finding novel regulators of non-centrosomal microtubule growth and assembly. Proteomic analyses of ncMTOC composition in differentiated cells are lacking, likely due to the enigmatic biochemical properties of ncMTOCs. To tackle this, we harnessed the unique advantages of enzyme-catalyzed proximity labeling (PL), which provides an in vivo 'snapshot' of protein associations. In PL, a labeling enzyme fused to a protein of interest catalyzes biotinylation of proteins lying within approximately 10 nm. The biotinylated proteins are subsequently harvested and identified by mass spectrometry. The original labeling enzyme BioID emerged as a revolutionary tool for studying proximal proteins in cultured cells; however, BioID was not adaptable to any living organisms. To overcome this problem, we developed the first demonstration of biotin-ligase based PL in C. elegans using the newly engineered PL enzymes TurboID and miniTurbo. Using this newly developed technology, we spatially restricted TurboID labeling activity to the apical ncMTOC by expressing TurboID fused to PTRN 1/CAMSAP in intestinal cells. We identified the proteins proximal to PTRN-1 with mass spectrometry and then validated the localization of top five proximal interactors using CRISPR/Cas-9 GFP knock-ins, all of which showed endogenous localization to the apical ncMTOC of intestinal cells. We focused on two hits: VAB-10B/MACF1, a spectraplakin, and WDR-62/H24G06.1, a previously uncharacterized protein which we identified as homologous to the primary microcephaly protein WDR62. VAB-10B and WDR-62 exhibited localization patterns very similar to PTRN-1 in intestinal cells and several other epithelial cell types. Using a tissue-specific protein degradation tool, we found that VAB-10B and WDR-62 regulate non centrosomal microtubule arrays likely through distinct mechanisms; loss of VAB-10B resulted in disorganized microtubules and delayed ɣ-TuRC localization, while loss of WDR-62 decreased microtubule numbers and abolished ɣ-TuRC localization. Depletion of VAB-10B and WDR-62 did not affect centrosomes, signifying their specificity to ncMTOCs. Their distinct mechanisms of action were further highlighted by their effects on dynamic microtubules; loss of VAB-10B did not change the overall number of dynamic microtubules but misplaced them from the apical surface, while loss of WDR-62 resulted in a significant loss of overall dynamic microtubules. These data support a model where two essential MTOC functions, microtubule growth and anchoring, are regulated by WDR-62 and VAB-10B, respectively. Additionally, VAB-10B and WDR-62 act downstream of cell polarity as the apical polarity protein PAR-3 properly localized following depletion of VAB-10B or WDR-62, and conversely depletion of PAR-3 mislocalized WDR-62. Finally, we found a critical role for actin in anchoring microtubules and speculate that this role could be downstream of VAB-10B and WDR-62. Together, this approach identified novel ncMTOC components and unveiled mechanisms critical for patterning exclusively non centrosomal microtubules. Additionally, this work uncovered the apical polarity complex as an upstream regulator of ncMTOCs. Overall, this work introduces a cutting-edge tool for identifying protein associations in living C. elegans, and additionally provides critical insight to in vivo regulation of MTOC activity, an understanding of which is imperative for tackling many human diseases.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2021; ©2021 |
| Publication date | 2021; 2021 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Sanchez, Ariana Danae |
|---|---|
| Degree supervisor | Feldman, Jessica L |
| Thesis advisor | Feldman, Jessica L |
| Thesis advisor | Cyert, Martha S, 1958- |
| Thesis advisor | Shen, Kang, 1972- |
| Thesis advisor | Stearns, Tim |
| Degree committee member | Cyert, Martha S, 1958- |
| Degree committee member | Shen, Kang, 1972- |
| Degree committee member | Stearns, Tim |
| Associated with | Stanford University, Department of Biology |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Ariana D. Sanchez. |
|---|---|
| Note | Submitted to the Department of Biology. |
| Thesis | Thesis Ph.D. Stanford University 2021. |
| Location | https://purl.stanford.edu/fj551ps3220 |
Access conditions
- Copyright
- © 2021 by Ariana Danae Sanchez
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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