Rapid turnover of stereocilia membrane proteins: evidence from the trafficking and mobility of plasma membrane Ca(2+)-ATPase 2.

M'hamed Grati, Mark E. Schneider, Karen Lipkow, Emanuel E. Strehler, Robert J. Wenthold, Bechara Kachar

Research output: Contribution to journalArticle

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Abstract

We studied the spatial distribution, mobility, and trafficking of plasma membrane Ca2+ATPase-2 (PMCA2), a protein enriched in the hair cell apical membrane and essential for hair cell function. Using immunofluorescence, we determined that PMCA2 is enriched in the stereocilia and present at a relatively low concentration in the kinocilium and in the remaining apical membrane. Using an antibody to the extracellular domain of PMCA2 as a probe, we observed that PMCA2 diffuses laterally from the stereocilia membrane and is internalized at the apical cell border maintaining an estimated half-life of residency in the stereocilia of approximately 5-7 h. A computer simulation of our data indicates that PMCA2 has an estimated global diffusion coefficient of 0.01-0.005 microm2/s. Using a green fluorescent protein tag, we observed that PMCA2 is rapidly delivered to the apical cell border from where it diffuses to the entire stereocilia surface. Fluorescence recovery after photobleaching experiments show that approximately 60% of PMCA2 in the stereocilia exhibit high mobility with a diffusion coefficient of 0.1-0.2 microm2/s, whereas the remaining pool represents a relatively immobile fraction. These results suggest that PMCA2 molecules maintain transient interactions with other components of the stereocilia, and the mobile pool of PMCA2 mediates the exchange between the stereocilia and the removal and delivery sites at the periphery of the apical cell surface. This rapid turnover of a major stereocilia membrane protein matches the previously described rapid turnover of proteins of the stereocilia actin core, further demonstrating that these organelles undergo rapid continuous renewal.

Original languageEnglish (US)
Pages (from-to)6386-6395
Number of pages10
JournalJournal of Neuroscience
Volume26
Issue number23
DOIs
StatePublished - Jun 7 2006
Externally publishedYes

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Stereocilia
Protein Transport
Calcium-Transporting ATPases
Adenosine Triphosphatases
Membrane Proteins
Cell Membrane
Fluorescence Recovery After Photobleaching
Membranes
Internship and Residency
Green Fluorescent Proteins
Organelles
Computer Simulation
Fluorescent Antibody Technique
Half-Life
Actins
Proteins

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Rapid turnover of stereocilia membrane proteins : evidence from the trafficking and mobility of plasma membrane Ca(2+)-ATPase 2. / Grati, M'hamed; Schneider, Mark E.; Lipkow, Karen; Strehler, Emanuel E.; Wenthold, Robert J.; Kachar, Bechara.

In: Journal of Neuroscience, Vol. 26, No. 23, 07.06.2006, p. 6386-6395.

Research output: Contribution to journalArticle

Grati, M'hamed ; Schneider, Mark E. ; Lipkow, Karen ; Strehler, Emanuel E. ; Wenthold, Robert J. ; Kachar, Bechara. / Rapid turnover of stereocilia membrane proteins : evidence from the trafficking and mobility of plasma membrane Ca(2+)-ATPase 2. In: Journal of Neuroscience. 2006 ; Vol. 26, No. 23. pp. 6386-6395.
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abstract = "We studied the spatial distribution, mobility, and trafficking of plasma membrane Ca2+ATPase-2 (PMCA2), a protein enriched in the hair cell apical membrane and essential for hair cell function. Using immunofluorescence, we determined that PMCA2 is enriched in the stereocilia and present at a relatively low concentration in the kinocilium and in the remaining apical membrane. Using an antibody to the extracellular domain of PMCA2 as a probe, we observed that PMCA2 diffuses laterally from the stereocilia membrane and is internalized at the apical cell border maintaining an estimated half-life of residency in the stereocilia of approximately 5-7 h. A computer simulation of our data indicates that PMCA2 has an estimated global diffusion coefficient of 0.01-0.005 microm2/s. Using a green fluorescent protein tag, we observed that PMCA2 is rapidly delivered to the apical cell border from where it diffuses to the entire stereocilia surface. Fluorescence recovery after photobleaching experiments show that approximately 60{\%} of PMCA2 in the stereocilia exhibit high mobility with a diffusion coefficient of 0.1-0.2 microm2/s, whereas the remaining pool represents a relatively immobile fraction. These results suggest that PMCA2 molecules maintain transient interactions with other components of the stereocilia, and the mobile pool of PMCA2 mediates the exchange between the stereocilia and the removal and delivery sites at the periphery of the apical cell surface. This rapid turnover of a major stereocilia membrane protein matches the previously described rapid turnover of proteins of the stereocilia actin core, further demonstrating that these organelles undergo rapid continuous renewal.",
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AB - We studied the spatial distribution, mobility, and trafficking of plasma membrane Ca2+ATPase-2 (PMCA2), a protein enriched in the hair cell apical membrane and essential for hair cell function. Using immunofluorescence, we determined that PMCA2 is enriched in the stereocilia and present at a relatively low concentration in the kinocilium and in the remaining apical membrane. Using an antibody to the extracellular domain of PMCA2 as a probe, we observed that PMCA2 diffuses laterally from the stereocilia membrane and is internalized at the apical cell border maintaining an estimated half-life of residency in the stereocilia of approximately 5-7 h. A computer simulation of our data indicates that PMCA2 has an estimated global diffusion coefficient of 0.01-0.005 microm2/s. Using a green fluorescent protein tag, we observed that PMCA2 is rapidly delivered to the apical cell border from where it diffuses to the entire stereocilia surface. Fluorescence recovery after photobleaching experiments show that approximately 60% of PMCA2 in the stereocilia exhibit high mobility with a diffusion coefficient of 0.1-0.2 microm2/s, whereas the remaining pool represents a relatively immobile fraction. These results suggest that PMCA2 molecules maintain transient interactions with other components of the stereocilia, and the mobile pool of PMCA2 mediates the exchange between the stereocilia and the removal and delivery sites at the periphery of the apical cell surface. This rapid turnover of a major stereocilia membrane protein matches the previously described rapid turnover of proteins of the stereocilia actin core, further demonstrating that these organelles undergo rapid continuous renewal.

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