Microinjection and localization of a 130K protein in living fibroblasts: a relationship to actin and fibronectin

doi:10.1016/S0092-8674(80)80035-3

Cell

Volume 19, Issue 3, March 1980, Pages 587-595

https://doi.org/10.1016/S0092-8674(80)80035-3 Get rights and content

Summary

A 130,000 dalton protein (130K protein), isolated from smooth muscle and having an as yet unknown function, has been studied through the use of microinjection of the protein covalently labeled with the fluorescent dye fluorescein isothiocyanate (FITC) into living cultured mammalian cells. The distribution of the 130K protein has also been investigated by several different methods including double label fluorescence microscopy of cells microinjected with FITC-130K protein and subsequently stained for 130K protein, actin or fibronectin by indirect immunofluorescence with rhodamine isothiocyanate (RITC)-labeled antibodies. Visualization of fluorescence in living fibroblasts 2–4 hr after microinjection of FITC-130K protein showed two major distributions of the fluorescent protein. One of these appeared as bright focal patches on the lower surface of the cells, and was similar to the distribution recently found by Geiger (1979) using immunofluorescent localization. Double label fluorescent microscopy of cells injected with FITC-130K protein and stained for actin 2 hr later by indirect immunofluorescence (RITC-antibodies) demonstrated that this focal distribution was at the ends of actin microfilament bundles. A second major distribution observed for the injected protein had a streaky or fibrillar appearance more commonly (but not always) located beneath the upper surface of the cell. Using antibodies against fibronectin and double label fluorescence microscopy, this second distribution of the injected protein was shown to correlate closely with the distribution of surface fibronectin on these cells. Where the fibronectin extended beyond a cell, the 130K protein remained strictly cellular and often appeared concentrated as if in an anchorage point. These findings suggest a possible role for the 130K protein in the organization of actin filaments at membrane attachment sites, and demonstrate that proteins of unknown function can be studied by the microinjection of their fluorescently labeled conjugates into living cells.

References (32)

AbercrombieM. et al.
The locomotion of fibroblasts. IV. Electron microscopy of the leading lamella
Exp. Cell Res.
(1971)
BlattlerD.P. et al.
quantitative electrophoresis in polyacrylamide gels of 2–40%
J. Chromatog.
(1972)
BurridgeK.
Direct identification of specific glycoproteins and antigens in sodium dodecyl sulfate gels
Methods in Enzymology
(1978)
GeigerB.
A 130K protein from chicken gizzard: its localization at the termini of microfilament bundles in cultured chicken cells
Cell
(1979)
HeaysmanJ.E.M. et al.
Early contacts between fibroblasts. An ultrastructural study
Exp. Cell Res.
(1973)
HynesR.O.
Role of surface alterations in cell transformation: the importance of proteases and surface proteins
Cell
(1974)
HynesR.O.
Cell surface proteins and malignant transformation
Biochim. Biophys. Acta
(1976)
HynesR.O. et al.
Relationships between fibronectin (LETS protein) and actin
Cell
(1978)
SingerI.I.
The fibronexus: a transmembrane association of fibronectin-containing fibers and bundles of 5 nm microfilaments in hamster and human fibroblasts
Cell
(1979)
VaheriA. et al.
High molecular weight, cell surface-associated glycoprotein (fibronectin) lost in malignant transformation
Biochim. Biophys. Acta
(1978)

BloseS.H.

Ten-nanometer filaments and mitosis: maintenance of structural continuity in dividing endothelial cells

BoltonA.E. et al.

The labelling of proteins to high specific radioactivities by conjugation to a ¹²⁵I-containing acylating agent

Biochem. J.

(1973)

BurridgeK.

Changes in cellular glycoproteins after transformation: identification of specific glycoproteins and antigens in sodium dodecylsulfate gels

ChenL.B. et al.

Correlation between tumor induction and cell surface LETS protein

FeramiscoJ.R.

Microinjection of fluorescently labeled α-actinin into living fibroblasts

FeramiscoJ.R. et al.

A rapid purification of α-actinin, filamin, and a 130,000-dalton protein from smooth muscle

J. Biol. Chem.

(1980)

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Cell

Microinjection and localization of a 130K protein in living fibroblasts: a relationship to actin and fibronectin

Summary

Exp. Cell Res.

J. Chromatog.

Methods in Enzymology

Cell

Exp. Cell Res.

Cell

Biochim. Biophys. Acta

Cell

Cell

Biochim. Biophys. Acta

Ten-nanometer filaments and mitosis: maintenance of structural continuity in dividing endothelial cells

The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent

Biochem. J.

Changes in cellular glycoproteins after transformation: identification of specific glycoproteins and antigens in sodium dodecylsulfate gels

Correlation between tumor induction and cell surface LETS protein

Microinjection of fluorescently labeled α-actinin into living fibroblasts

A rapid purification of α-actinin, filamin, and a 130,000-dalton protein from smooth muscle

J. Biol. Chem.

The labelling of proteins to high specific radioactivities by conjugation to a ¹²⁵I-containing acylating agent