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Microtubules and microfilaments during cell spreading and colony formation in PK 15 epithelial cells
Abstract:
We have studied the distribution of microtubules and microfilaments during the cell spreading and subsequent colony formation in PK 15 pig kidney epithelial cells using indirect immunofluorescence. During the cell spreading on a solid substratum, microtubules grew out from the region around the nucleus, and a collar of microfilament bundles formed around the cell periphery. Although virtually all well-spread cells showed a complex microtubular network, distinctly different patterns of stress fibers were observed. In small colonies, the most commonly observed pattern was a ring of microfilament bundles that appeared to be in register between adjacent cells and encircled the entire colony in a fashion similar to that seen in single cells. In large colonies (more than 50 cells), approximately 60% of the cells displayed clearly stained microfilament bundles, either at the cell periphery or throughout their cytoplasm, whereas in the remaining 40%, no microfilament bundles were observed and only the outline of the cells was delineated by interaction with anti-actin. Such "negative" cells were seen in groups alongside "positive" cells (i.e., cells possessing extensive stress fiber networks) within the same colony. Independent of their stress fiber phenotype, all cells maintained a flattened shape and an extensive network of microtubules. We suggest that dense microfilament bundles are not a uniform feature of well-spread PI 15 cells in culture and that a loss of microfilament bundle occurs in some cells.
Insights
Microtubules and microfilaments in pig kidney cells show distinct patterns during spreading and colony formation. Not all well-spread cells uniformly display dense microfilament bundles, suggesting a loss can occur.
Area of Science:
- Cell Biology
- Cytoskeleton Dynamics
Background:
- Cell spreading and colony formation are critical processes in epithelial tissue development.
- The cytoskeleton, composed of microtubules and microfilaments, plays a vital role in cell shape, motility, and organization.
Purpose of the Study:
- To investigate the distribution and patterns of microtubules and microfilaments during cell spreading and colony formation in PK 15 pig kidney epithelial cells.
- To identify variations in cytoskeletal organization, particularly stress fibers, within cell colonies.
Main Methods:
- Indirect immunofluorescence was used to visualize microtubules and microfilaments.
- PK 15 cells were cultured on a solid substratum to observe cell spreading and colony formation.
- Staining with anti-actin antibodies identified microfilament bundles.
Main Results:
- During cell spreading, microtubules radiated from the nucleus, and microfilaments formed a peripheral collar.
- In small colonies, microfilament bundles formed a continuous ring around adjacent cells.
- In large colonies, approximately 60% of cells had visible microfilament bundles, while 40% lacked them, showing only cell outlines.
- Both 'positive' (with stress fibers) and 'negative' (without stress fibers) cells coexisted within colonies.
- All cells, regardless of stress fiber presence, maintained a flattened shape and microtubule network.
Conclusions:
- Dense microfilament bundles are not a universal feature of all well-spread PK 15 cells in culture.
- A loss or absence of microfilament bundles can occur in a subset of cells within a colony.
- Microtubule networks remain extensive and stable irrespective of the microfilament bundle phenotype.