Related Experiment Videos
Human spectrin Src homology 3 domain binding protein 1 regulates macropinocytosis in NIH 3T3 cells
1Laboratory of Molecular Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA.
Abstract:
Macropinocytosis is an endocytic process that occurs through non-clathrin coated vesicles larger than 0.2 microm in diameter. Although macropinocytic vesicles are readily visualized in cultured cells by the introduction of fluorescent, water-soluble dyes into the culture medium, protein markers associated with this type of vesicles have not yet been well defined. Here, we report that human spectrin SH3 domain binding protein 1, or Hssh3bp1, associates with macropinosomes in NIH 3T3 fibroblasts. Hssh3bp1 macropinosomes are heterogeneous in morphology and size, do not endocytose transferrin and are resistant to brefeldin A treatment. Cytochalasin D, and wortmannin block endocytosis of fluorescent dyes into the Hssh3bp1 macropinosomes and dramatically affect their morphology. Overexpression of Hssh3bp1-green fluorescent protein abolished fusion of vesicles resulting in a decreased endocytosis of fluorescence dyes, thus suggesting a potential regulatory role of Hssh3bp1 in macropinocytosis. In the macropinosomes of NIH 3T3 cells, Hssh3bp1 associates with a 200-kDa protein that crossreacts with a monoclonal antibody to the erythroid alpha-spectrin SH3 domain. Thus macropinosomes in cells may contain a spectrin-like protein.
Insights
Human spectrin SH3 domain binding protein 1 (Hssh3bp1) associates with macropinosomes, key cellular structures for large vesicle formation. This finding suggests Hssh3bp1 may regulate macropinocytosis, a vital cellular process.
Area of Science:
- Cell Biology
- Molecular Biology
- Endocytosis
Background:
- Macropinocytosis is a cellular uptake mechanism forming large vesicles (>0.2 µm).
- Protein markers for macropinosomes remain poorly defined.
- Identifying these markers is crucial for understanding macropinocytosis regulation.
Purpose of the Study:
- To identify novel protein markers associated with macropinosomes.
- To investigate the role of human spectrin SH3 domain binding protein 1 (Hssh3bp1) in macropinocytosis.
Main Methods:
- Utilized NIH 3T3 fibroblasts and fluorescent dyes to visualize macropinosomes.
- Investigated Hssh3bp1 localization and association within macropinosomes.
- Examined the effects of pharmacological inhibitors (cytochalasin D, wortmannin) and Hssh3bp1 overexpression on macropinocytosis.
Main Results:
- Hssh3bp1 was identified as a novel protein marker associating with macropinosomes.
- Hssh3bp1-positive macropinosomes exhibited distinct characteristics (heterogeneous size, resistance to brefeldin A, lack of transferrin uptake).
- Inhibition of actin dynamics and PI3K signaling affected Hssh3bp1 macropinosomes; Hssh3bp1 overexpression impaired vesicle fusion and dye uptake.
Conclusions:
- Hssh3bp1 is a novel component of macropinosomes in NIH 3T3 cells.
- Hssh3bp1 plays a regulatory role in macropinocytosis, potentially influencing vesicle fusion and cargo internalization.
- Macropinosomes may contain spectrin-like proteins, including Hssh3bp1.