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Assay for early cytoplasmic effects of the src gene product of Rous sarcoma virus
Abstract:
When microinjected into normal fibroblasts, cytoplasmic extracts of cells transformed by Rous sarcoma virus caused dissolution of microfilament bundles. This activity was not found in extracts of normal cells. The maximum effect was seen within 30 min of injection, and the activity could still be measured after a 10-fold dilution of the cytoplasmic extracts (14 mg/ml original protein concentration). The activity was trypsin sensitive and was destroyed by boiling, but was not RNase sensitive. Protein synthesis was not required for the disruption of actin-containing stress fibers by the injected activity. Microinjected cytoplasts prepared from normal 3T3 cells also showed dissolution of microfilament bundles, indicating that the cell nucleus was not required for expression of activity. Extracts made from fibroblasts transformed by Rous sarcoma virus having a temperature-sensitive mutation in the src gene were also temperature sensitive in the microinjection assay. Thus, the activity of extracts from cells infected with src mutant virus, but not from cells infected with wild-type virus, was destroyed either by in vitro incubation of the extract at the nonpermissive temperature before injection or by incubation of recipient cells at the nonpermissive temperature after injection. We conclude that the microinjection assay can detect a cytoplasmic activity coded for by the src gene of Rous sarcoma virus and that an early direct or indirect target of the src gene product is the cytoskeleton and cell motility system. This result is discussed in relation to the hypothesis that submembranous arrays of microfilaments, microtubules, and their associated proteins interact with cell surface receptors to form a surface modulating assembly that functions as a key regulator of cell growth.
Insights
Cytoplasmic extracts from Rous sarcoma virus-transformed cells disrupt microfilament bundles in normal fibroblasts. This activity, linked to the src gene, targets the cytoskeleton and cell motility.
Area of Science:
- Cell Biology
- Virology
- Molecular Biology
Background:
- Rous sarcoma virus (RSV) transformation alters cellular morphology and function.
- The src gene product is implicated in these transformations, but its direct targets remain under investigation.
- Cytoskeletal organization is crucial for cell shape, motility, and growth regulation.
Purpose of the Study:
- To identify and characterize cytoplasmic activities in RSV-transformed cells that affect the cytoskeleton.
- To determine if these activities are directly or indirectly mediated by the src gene product.
- To investigate the role of the src gene in cytoskeletal regulation and cell growth control.
Main Methods:
- Microinjection of cytoplasmic extracts from RSV-transformed and normal fibroblasts into recipient normal fibroblasts.
- Assay for the dissolution of actin-containing microfilament bundles (stress fibers) post-injection.
- Use of temperature-sensitive src mutant RSV to assess the temperature sensitivity of the observed activity.
- Treatment of extracts and cells with enzymes (trypsin, RNase) and heat to characterize the active component.
Main Results:
- Cytoplasmic extracts from RSV-transformed cells, but not normal cells, induced rapid dissolution of microfilament bundles upon microinjection.
- The activity was trypsin-sensitive, heat-labile, and not dependent on protein synthesis or the cell nucleus.
- Extracts from cells infected with a temperature-sensitive src mutant showed temperature-dependent activity, correlating the effect with the src gene product.
- The observed activity was dose-dependent and stable to a 10-fold dilution.
Conclusions:
- A cytoplasmic activity encoded by the Rous sarcoma virus src gene directly or indirectly targets the cell's cytoskeleton and motility system.
- This src gene-mediated cytoskeletal disruption is an early event in viral transformation.
- The findings support the hypothesis that src-induced alterations in the cytoskeleton-associated surface modulating assembly regulate cell growth.