Striated flagellar roots

Isolation and partial characterization of a calcium-modulated contractile organelle

Jeffrey L Salisbury, A. Baron, B. Surek, M. Melkonian

Research output: Contribution to journalArticle

269 Citations (Scopus)

Abstract

We report the isolation of striated flagellar roots from the Prasinophycean green alga Tetraselmis striata using sedimentation in gradients of sucrose and flotation on gradients of colloidal silica. PAGE in the presence of 0.1% SDS demonstrates that striated flagellar roots are composed of a number of polypeptides, the most predominant one being a protein of 20,000 M(r). The 20,000 M(r) protein band represents ~63% of the Coomassie Brilliant Blue staining of gels of isolated flagellar roots. Two-dimensional gel electrophoresis (isoelectric focusing and SDS PAGE) resolves the major 20,000 M(r) flagellar root protein into two components of nearly identical M(r), but of differing isoelectric points (i.e., pI's of 4.9 and 4.8), which we have designated 20,000 M(r)-α and 20,000-M(r)-β, respectively. Densitometric scans of two-dimensional gels of cell extracts indicate that the 20,000-M(r)-α and -β polypeptides vary, in their stoichiometry, between 2:1 and 1:1. This variability appears to be related to the state of contraction or extension of the striated flagellar roots at the time of cell lysis. Incubation of cells with 32PO4 followed by analysis of cell extracts by two-dimensional gel electrophoresis and autoradiography reveals that the more acidic 20,000-M(r)-β component is phosphorylated and the 20,000-M(r)-α component contains no detectable label. These results suggest that the 20,000-M(r)-α component is converted to the more acidic 20,000-M(r)-β form by phosphorylation. Both the 20,000-M(r)-α and -β flagellar root components exhibit a calcium-induced reduction in relative electrophoretic mobilities in two-dimensional alkaline urea gels. Antiserum raised in rabbits against the 20,000-M(r) protein binds to both the 20,000-M(r)-α and 20,000-M(r)-β forms of the flagellar root protein when analyzed by electrophoretic immunoblot techniques. Indirect immunofluorescence on vegetative or interphase cells demonstrate that the antibodies bind to two cyclindrical organelles located in the anterior region of the cell. Immunocytochemical investigations at ultrastructural resolution using this antiserum and a colloidal gold-conjugated antirabbit-IgG reveals immunospecific labeling of striated flagellar roots and their extensions. We conclude that striated flagellar roots are simple ion-sensitive contractile organelles composed predominantly of a 20,000 M(r) calcium-binding phosphoprotein, and that this protein is largely responsible for the motile behavior of these organelles.

Original languageEnglish (US)
Pages (from-to)962-970
Number of pages9
JournalJournal of Cell Biology
Volume99
Issue number3
StatePublished - 1984
Externally publishedYes

Fingerprint

Myeloma Proteins
Organelles
Calcium
Gels
Electrophoresis, Gel, Two-Dimensional
Cell Extracts
Immune Sera
Proteins
Connectin
Gold Colloid
Peptides
Chlorophyta
Calcium-Binding Proteins
Phosphoproteins
Interphase
Isoelectric Point
Isoelectric Focusing
Indirect Fluorescent Antibody Technique
Autoradiography
Silicon Dioxide

ASJC Scopus subject areas

  • Cell Biology

Cite this

Striated flagellar roots : Isolation and partial characterization of a calcium-modulated contractile organelle. / Salisbury, Jeffrey L; Baron, A.; Surek, B.; Melkonian, M.

In: Journal of Cell Biology, Vol. 99, No. 3, 1984, p. 962-970.

Research output: Contribution to journalArticle

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abstract = "We report the isolation of striated flagellar roots from the Prasinophycean green alga Tetraselmis striata using sedimentation in gradients of sucrose and flotation on gradients of colloidal silica. PAGE in the presence of 0.1{\%} SDS demonstrates that striated flagellar roots are composed of a number of polypeptides, the most predominant one being a protein of 20,000 M(r). The 20,000 M(r) protein band represents ~63{\%} of the Coomassie Brilliant Blue staining of gels of isolated flagellar roots. Two-dimensional gel electrophoresis (isoelectric focusing and SDS PAGE) resolves the major 20,000 M(r) flagellar root protein into two components of nearly identical M(r), but of differing isoelectric points (i.e., pI's of 4.9 and 4.8), which we have designated 20,000 M(r)-α and 20,000-M(r)-β, respectively. Densitometric scans of two-dimensional gels of cell extracts indicate that the 20,000-M(r)-α and -β polypeptides vary, in their stoichiometry, between 2:1 and 1:1. This variability appears to be related to the state of contraction or extension of the striated flagellar roots at the time of cell lysis. Incubation of cells with 32PO4 followed by analysis of cell extracts by two-dimensional gel electrophoresis and autoradiography reveals that the more acidic 20,000-M(r)-β component is phosphorylated and the 20,000-M(r)-α component contains no detectable label. These results suggest that the 20,000-M(r)-α component is converted to the more acidic 20,000-M(r)-β form by phosphorylation. Both the 20,000-M(r)-α and -β flagellar root components exhibit a calcium-induced reduction in relative electrophoretic mobilities in two-dimensional alkaline urea gels. Antiserum raised in rabbits against the 20,000-M(r) protein binds to both the 20,000-M(r)-α and 20,000-M(r)-β forms of the flagellar root protein when analyzed by electrophoretic immunoblot techniques. Indirect immunofluorescence on vegetative or interphase cells demonstrate that the antibodies bind to two cyclindrical organelles located in the anterior region of the cell. Immunocytochemical investigations at ultrastructural resolution using this antiserum and a colloidal gold-conjugated antirabbit-IgG reveals immunospecific labeling of striated flagellar roots and their extensions. We conclude that striated flagellar roots are simple ion-sensitive contractile organelles composed predominantly of a 20,000 M(r) calcium-binding phosphoprotein, and that this protein is largely responsible for the motile behavior of these organelles.",
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N2 - We report the isolation of striated flagellar roots from the Prasinophycean green alga Tetraselmis striata using sedimentation in gradients of sucrose and flotation on gradients of colloidal silica. PAGE in the presence of 0.1% SDS demonstrates that striated flagellar roots are composed of a number of polypeptides, the most predominant one being a protein of 20,000 M(r). The 20,000 M(r) protein band represents ~63% of the Coomassie Brilliant Blue staining of gels of isolated flagellar roots. Two-dimensional gel electrophoresis (isoelectric focusing and SDS PAGE) resolves the major 20,000 M(r) flagellar root protein into two components of nearly identical M(r), but of differing isoelectric points (i.e., pI's of 4.9 and 4.8), which we have designated 20,000 M(r)-α and 20,000-M(r)-β, respectively. Densitometric scans of two-dimensional gels of cell extracts indicate that the 20,000-M(r)-α and -β polypeptides vary, in their stoichiometry, between 2:1 and 1:1. This variability appears to be related to the state of contraction or extension of the striated flagellar roots at the time of cell lysis. Incubation of cells with 32PO4 followed by analysis of cell extracts by two-dimensional gel electrophoresis and autoradiography reveals that the more acidic 20,000-M(r)-β component is phosphorylated and the 20,000-M(r)-α component contains no detectable label. These results suggest that the 20,000-M(r)-α component is converted to the more acidic 20,000-M(r)-β form by phosphorylation. Both the 20,000-M(r)-α and -β flagellar root components exhibit a calcium-induced reduction in relative electrophoretic mobilities in two-dimensional alkaline urea gels. Antiserum raised in rabbits against the 20,000-M(r) protein binds to both the 20,000-M(r)-α and 20,000-M(r)-β forms of the flagellar root protein when analyzed by electrophoretic immunoblot techniques. Indirect immunofluorescence on vegetative or interphase cells demonstrate that the antibodies bind to two cyclindrical organelles located in the anterior region of the cell. Immunocytochemical investigations at ultrastructural resolution using this antiserum and a colloidal gold-conjugated antirabbit-IgG reveals immunospecific labeling of striated flagellar roots and their extensions. We conclude that striated flagellar roots are simple ion-sensitive contractile organelles composed predominantly of a 20,000 M(r) calcium-binding phosphoprotein, and that this protein is largely responsible for the motile behavior of these organelles.

AB - We report the isolation of striated flagellar roots from the Prasinophycean green alga Tetraselmis striata using sedimentation in gradients of sucrose and flotation on gradients of colloidal silica. PAGE in the presence of 0.1% SDS demonstrates that striated flagellar roots are composed of a number of polypeptides, the most predominant one being a protein of 20,000 M(r). The 20,000 M(r) protein band represents ~63% of the Coomassie Brilliant Blue staining of gels of isolated flagellar roots. Two-dimensional gel electrophoresis (isoelectric focusing and SDS PAGE) resolves the major 20,000 M(r) flagellar root protein into two components of nearly identical M(r), but of differing isoelectric points (i.e., pI's of 4.9 and 4.8), which we have designated 20,000 M(r)-α and 20,000-M(r)-β, respectively. Densitometric scans of two-dimensional gels of cell extracts indicate that the 20,000-M(r)-α and -β polypeptides vary, in their stoichiometry, between 2:1 and 1:1. This variability appears to be related to the state of contraction or extension of the striated flagellar roots at the time of cell lysis. Incubation of cells with 32PO4 followed by analysis of cell extracts by two-dimensional gel electrophoresis and autoradiography reveals that the more acidic 20,000-M(r)-β component is phosphorylated and the 20,000-M(r)-α component contains no detectable label. These results suggest that the 20,000-M(r)-α component is converted to the more acidic 20,000-M(r)-β form by phosphorylation. Both the 20,000-M(r)-α and -β flagellar root components exhibit a calcium-induced reduction in relative electrophoretic mobilities in two-dimensional alkaline urea gels. Antiserum raised in rabbits against the 20,000-M(r) protein binds to both the 20,000-M(r)-α and 20,000-M(r)-β forms of the flagellar root protein when analyzed by electrophoretic immunoblot techniques. Indirect immunofluorescence on vegetative or interphase cells demonstrate that the antibodies bind to two cyclindrical organelles located in the anterior region of the cell. Immunocytochemical investigations at ultrastructural resolution using this antiserum and a colloidal gold-conjugated antirabbit-IgG reveals immunospecific labeling of striated flagellar roots and their extensions. We conclude that striated flagellar roots are simple ion-sensitive contractile organelles composed predominantly of a 20,000 M(r) calcium-binding phosphoprotein, and that this protein is largely responsible for the motile behavior of these organelles.

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