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Coordination between actin and microtubules is important for numerous cellular processes in diverse eukaryotes. In plants, tip-growing cells require actin for cell expansion and microtubules for orientation of cell expansion, but how the two cytoskeletons are linked is an open question. In tip-growing cells of the moss Physcomitrella patens , we show that an actin cluster near the cell apex dictates the direction of rapid cell expansion. Formation of this structure depends on the convergence of microtubules near the cell tip. We discovered that microtubule convergence requires class VIII myosin function, and actin is necessary for myosin VIII–mediated focusing of microtubules. The loss of myosin VIII function affects both networks, indicating functional connections among the three cytoskeletal components. Our data suggest that microtubules direct localization of formins, actin nucleation factors, that generate actin filaments further focusing microtubules, thereby establishing a positive feedback loop ensuring that actin polymerization and cell expansion occur at a defined site resulting in persistent polarized growth.
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Effective and efficient cytoskeleton (actin and microtubules) fluorescence staining of adherent eukaryotic cells v2
Eukaryotic microbes, protists, are highly diverse organisms with complex cytoskeletal elements used for movement consisting mostly of actin-myosin and microtubules. In order to visualize the cytoskeletal elements researchers may take a microscopical approach based on immunocytochemistry. Presented here is an efficient and effective for staining and visualizing actin microfilaments stained with phalloidin, nuclei stained with Hoechst 33342, and microtubules labeled using an alpha tubulin antibody. This protocol was developed for amoeboid protists, but will likely work on other adherent eukaryotic cells. Protocol is adapted from the following citations.
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- Award ID(s):
- 2100888
- PAR ID:
- 10574816
- Publisher / Repository:
- Springer Nature
- Date Published:
- Format(s):
- Medium: X
- Institution:
- Protocols.io
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Posted Content:
- https://doi.org/10.17504/protocols.io.kxygxeerzv8j/v2
