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Updated: Aug 14, 2026
Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids
Published on: December 13, 2017
Identification and organization of the components in the isolated microvillus cytoskeleton
Abstract:
We have examined the effects of ATP and deoxycholate (DOC) on the cytoskeletal organization of Triton-demembranated microvilli (MV) isolated from chicken intestine brush borders. Isolated MV are composed of a core of tightly bundled microfilaments from which arms project laterally to the plasma membrane with a 33-nm periodicity. These lateral arms spiral around the core microfilaments as a helix with a 25 degrees pitch. Demembranated MV consist of four polypeptides with mol wt of 110,000, 95,000, 68,000, and 42,000, present in molar ratios of 1.1:1.6:1.3:10.0. After addition of 50 microM ATP and 0.1 mM Mg++, the cytoskeletons are organized as a tight bundle of microfilaments from which lateral arms are missing. In these ATP-treated cytoskeletons, the 110-kdalton polypeptide is reduced in amount and the 95,000, 68,000, and 42,000 polypeptides are present in a 1.3:1.2:10.0 ratio. In contrast, after incubation with 0.5% DOC, the core microfilaments are no longer tightly bundled yet the lateral arms remain attached with a distinct 33-nm periodicity. These DOC-treated cytoskeletons are depleted of the 95,000 and 68,000 polypeptides and are composed of the 110,000 and 42,000 polypeptides in a 2:10 molar ratio. These results suggest that the microfilaments are associated into a core bundle by the 95- and 68-kdalton polypeptides and from this core bundle project the lateral arms composed of the 110-kdalton polypeptide.
Insights
Adenosine triphosphate (ATP) and deoxycholate (DOC) differentially affect chicken intestinal microvilli (MV) cytoskeleton organization. ATP treatment disassembles lateral arms, while DOC treatment disrupts microfilament bundling, revealing protein functions in MV structure.
Area of Science:
- Cell Biology
- Cytoskeletal Dynamics
- Protein Biochemistry
Background:
- Microvilli (MV) are essential cellular projections with a core of actin microfilaments and associated proteins.
- The precise organization and protein composition of the MV cytoskeleton are crucial for their function.
- Understanding how MV cytoskeletal components interact is key to deciphering cellular structure and motility.
Purpose of the Study:
- To investigate the roles of specific polypeptides in the structural organization of chicken intestinal microvilli (MV).
- To determine the effects of adenosine triphosphate (ATP) and deoxycholate (DOC) on MV cytoskeletal organization and protein composition.
- To elucidate the protein associations responsible for microfilament bundling and lateral arm formation in MV.
Main Methods:
- Isolation of Triton-demembranated microvilli (MV) from chicken intestine brush borders.
- Biochemical analysis of MV polypeptide composition using SDS-PAGE and molar ratio determination.
- Treatment of demembranated MV with ATP and deoxycholate (DOC) to observe cytoskeletal structural changes via electron microscopy and protein analysis.
Main Results:
- ATP treatment (with Mg++) caused the loss of lateral arms and reduced the 110-kdalton polypeptide, altering the ratios of remaining proteins.
- Deoxycholate (DOC) treatment dispersed the microfilament core but preserved lateral arms, depleting 95- and 68-kdalton polypeptides.
- DOC-treated MV retained the 110-kdalton and 42,000-kdalton polypeptides in a 2:10 molar ratio, suggesting their role in lateral arm structure.
Conclusions:
- The 95-kdalton and 68-kdalton polypeptides are essential for associating microfilaments into the core bundle of MV.
- The 110-kdalton polypeptide is a major component of the lateral arms projecting from the MV core.
- These findings delineate specific protein functions in maintaining the structural integrity and organization of intestinal microvilli.
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