UNLABELLED: Adeno-associated virus was used to transduce primary mouse osteoclasts with the B1 isoform of vacuolar H(+)-ATPase. B1, which is not normally expressed in osteoclasts, was correctly targeted to ruffled membranes of resorbing osteoclasts. Mutant subunit B1 that lacked a functional actin-binding site did not accumulate in ruffled membranes. INTRODUCTION: The B1 "kidney" and B2 "brain" isoforms of subunit B of vacuolar H(+)-ATPase (V-ATPase) have actin binding sites that mediate interactions between the intact enzyme and filamentous-actin. Accumulating data support the hypothesis that the actin binding activity in subunit B is required for targeting of V-ATPases to the ruffled plasma membrane of osteoclasts. This study was designed to directly test this hypothesis. MATERIALS AND METHODS: Osteoclasts express B2, but not B1. Adeno-associated virus vectors were used to transduce mouse osteoclasts with wildtype B1 or B1(mut), a full-length B subunit that contained minor alterations that disrupted actin-binding activity. Immunofluorescence was performed using polyclonal antibodies specific for subunit E, B2, and B1 of V-ATPase. Immunoprecipitations were performed using an anti-E subunit antibody. Microfilaments were detected with phalloidin and actin rings were stained with phalloidin or anti-vinculin antibodies. Images were collected using a confocal microscope. RESULTS: Immunoprecipitations of transduced osteoclasts suggested that both B1 and B1(mut) assembled with endogenous V-ATPase subunits to form intact enzyme in osteoclasts. Both B1 and B1(mut) were localized like endogenous V-ATPase subunits in unactivated osteoclasts. Wildtype B1 associated with the detergent-insoluble cytoskeleton and was transported to ruffled membranes of resorbing osteoclasts. In contrast, B1(mut) failed to associate with the actin cytoskeleton and was not transported efficiently to ruffled membranes. CONCLUSIONS: The B1 isoform of B subunit contains the necessary information for targeting to the ruffled membranes of osteoclasts even though it is not normally expressed in osteoclasts. The actin binding activity of B1 is involved in proper ruffled membrane targeting.
UNLABELLED: Adeno-associated virus was used to transduce primary mouse osteoclasts with the B1 isoform of vacuolar H(+)-ATPase. B1, which is not normally expressed in osteoclasts, was correctly targeted to ruffled membranes of resorbing osteoclasts. Mutant subunit B1 that lacked a functional actin-binding site did not accumulate in ruffled membranes. INTRODUCTION: The B1 "kidney" and B2 "brain" isoforms of subunit B of vacuolar H(+)-ATPase (V-ATPase) have actin binding sites that mediate interactions between the intact enzyme and filamentous-actin. Accumulating data support the hypothesis that the actin binding activity in subunit B is required for targeting of V-ATPases to the ruffled plasma membrane of osteoclasts. This study was designed to directly test this hypothesis. MATERIALS AND METHODS: Osteoclasts express B2, but not B1. Adeno-associated virus vectors were used to transduce mouse osteoclasts with wildtype B1 or B1(mut), a full-length B subunit that contained minor alterations that disrupted actin-binding activity. Immunofluorescence was performed using polyclonal antibodies specific for subunit E, B2, and B1 of V-ATPase. Immunoprecipitations were performed using an anti-E subunit antibody. Microfilaments were detected with phalloidin and actin rings were stained with phalloidin or anti-vinculin antibodies. Images were collected using a confocal microscope. RESULTS: Immunoprecipitations of transduced osteoclasts suggested that both B1 and B1(mut) assembled with endogenous V-ATPase subunits to form intact enzyme in osteoclasts. Both B1 and B1(mut) were localized like endogenous V-ATPase subunits in unactivated osteoclasts. Wildtype B1 associated with the detergent-insoluble cytoskeleton and was transported to ruffled membranes of resorbing osteoclasts. In contrast, B1(mut) failed to associate with the actin cytoskeleton and was not transported efficiently to ruffled membranes. CONCLUSIONS: The B1 isoform of B subunit contains the necessary information for targeting to the ruffled membranes of osteoclasts even though it is not normally expressed in osteoclasts. The actin binding activity of B1 is involved in proper ruffled membrane targeting.
Authors: Edgardo J Toro; Jian Zuo; David A Ostrov; Dana Catalfamo; Vivian Bradaschia-Correa; Victor Arana-Chavez; Aliana R Caridad; John K Neubert; Thomas J Wronski; Shannon M Wallet; L Shannon Holliday Journal: J Biol Chem Date: 2012-04-02 Impact factor: 5.157
Authors: E J Toro; J Zuo; A Gutierrez; A Guiterrez; R L La Rosa; A J Gawron; V Bradaschia-Correa; V Arana-Chavez; C Dolce; M F Rivera; L Kesavalu; I Bhattacharyya; J K Neubert; L S Holliday Journal: J Dent Res Date: 2013-08-19 Impact factor: 6.116
Authors: Eric M Serrano; Ryan D Ricofort; Jian Zuo; Noelle Ochotny; Morris F Manolson; L Shannon Holliday Journal: Biochem Biophys Res Commun Date: 2009-08-26 Impact factor: 3.575
Authors: David A Ostrov; Andrew T Magis; Thomas J Wronski; Edward K L Chan; Edgardo J Toro; Richard E Donatelli; Kristen Sajek; Ireni N Haroun; Michael I Nagib; Ana Piedrahita; Ashley Harris; L Shannon Holliday Journal: J Med Chem Date: 2009-08-27 Impact factor: 7.446