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The cytoskeleton and cell volume regulation
Abstracts / Comparative Biochemistry and Physiology, Part A 126 (2000) S1-S163
S 105
THE CYTOSKELETON AND CELL VOLUME REGULATION
Mills J.W. Division of Health Sciences, Clarkson University, Potsdam, NY 13699 The cytoskeleton has been implicated in playing a role in the mechanism of cell volume control especially with regard to the changes in ion transport that occur in cells subjected to hypotonic media (the RVD response) or hypertonic media (the RVI response). The component of the cytoskeleton most often cited as a participant in the volume control process is that group of proteins belonging to the broadly defined actin filament system (F-actin). Four possible roles for F-actin have been proposed : 1. As a resistive force for prevention of membrane distention, 2. As a structural framework or active participant in the movement of membrane transporters to and from the cell membrane, 3. As a structural component for positioning and maintaining the transporters in the appropriate membrane domain. 4. As a regulatory protein that is part of tile signalling mechanism or directly alters transporter function. The evidence to support these roles come from studies on diverse cell types using three basic approaches. The first is simple observation and correlation of cytoskeletal change within the time frame of regulatory volume changes. The second is by treatment with inhibitors of F-actin dynamics or steady-state organization. The third is by molecular modification of the cytoskeletal proteins. Each has produced valuable insights but need to be used with caution because of inherent problems in use. In the case of RVD significant data indicates that disruption of the F-actin cytoskeleton leads to altered function of either a C1 channel or, in epithelia, of the Na+,K+,2C1- co-transporter. In RVI there is strong evidence that F-actin, in combination with myosin II, plays a role in the activity of the co-transporter. However, some studies indicate that the changes in F-actin are not a key to changes in ion transport but, rather, are a direct result of the alterations in ion flux or content. Finally, not all cell types respond the same way to cytoskeletal perturbation, indicating that a role for the cytoskeleton may be cell specific rather than fundamental.
STUDY OF SOME M E C H A N I S M S CONTROLLING THE BETAINE UPTAKE CAPACITY OF THE G R A M POSITIVE BACTERIA L A C T O C O C C U S LACTIS NCDO763 Obis D., Guillot A. and Mistou M-Y. Unit6 de Recherches Biochimie et Structure des Prot6ines, INRA, 78352 Jouy-en-Josas cedex, France In L. lactis NCDO763, the activity of an unique ABC transport system called BusA or OpuA is responsible for osmodependent betaine accumulation.~'~'3 BusA is a close relative of OpuA, its functional homolog in B. subtilis, 4 but the lactococcal transport system presents an original fusion event between the membrane and the substrate- binding domains. As in E. coli or B. subtilis, osmotic upshock increases the betaine uptake capacity of L. lactis by raising the transcription of busA and the activity of BusA. Our present work focuses on the mechanisms controlling the osmotic induction of busA. The transcriptional start sites of busA were mapped and two promoters were found to control the expression of busA The distal promoter (PI) presents a consensus -35 region and an extented -10 box : it is silent at low osmolarity and activated above 0.4 osM. A second, proximal promoter (P2) seems to be responsible of a low, basal level of transcription indifferent to the medium osmolarity. The level of luciferase activity in strains expressing chromosomal busA:luxAluxB fusions were measured during growth under osmotic constrainst to specify the regulatory regions necessary for the osmotic induction. These constructs were also used to study the role of compatible solute accumulation on the down-regulation of busA transcription. In L. lactis NCDO763, a gene located upstream of busA codes for a transcriptional regulator of the GntR family. In vivo experiments indicated that this protein acts as a repressor of busA expression. I. 2. 3. 4.
Obis, D. et al. (1999) J. Bacteriol. 181 6238-6246 van der Heide, T. and B. Poolman (2000) J. Bacteriol. 182 203-206 Bouvier, J. et al. (2000) J. Mol. Microbiol. Biotechnol. 2 199-205 Kempf, B. and E. Bremer (1995) J. Biol Chem. 270 16701-13
S 105
THE CYTOSKELETON AND CELL VOLUME REGULATION
Mills J.W. Division of Health Sciences, Clarkson University, Potsdam, NY 13699 The cytoskeleton has been implicated in playing a role in the mechanism of cell volume control especially with regard to the changes in ion transport that occur in cells subjected to hypotonic media (the RVD response) or hypertonic media (the RVI response). The component of the cytoskeleton most often cited as a participant in the volume control process is that group of proteins belonging to the broadly defined actin filament system (F-actin). Four possible roles for F-actin have been proposed : 1. As a resistive force for prevention of membrane distention, 2. As a structural framework or active participant in the movement of membrane transporters to and from the cell membrane, 3. As a structural component for positioning and maintaining the transporters in the appropriate membrane domain. 4. As a regulatory protein that is part of tile signalling mechanism or directly alters transporter function. The evidence to support these roles come from studies on diverse cell types using three basic approaches. The first is simple observation and correlation of cytoskeletal change within the time frame of regulatory volume changes. The second is by treatment with inhibitors of F-actin dynamics or steady-state organization. The third is by molecular modification of the cytoskeletal proteins. Each has produced valuable insights but need to be used with caution because of inherent problems in use. In the case of RVD significant data indicates that disruption of the F-actin cytoskeleton leads to altered function of either a C1 channel or, in epithelia, of the Na+,K+,2C1- co-transporter. In RVI there is strong evidence that F-actin, in combination with myosin II, plays a role in the activity of the co-transporter. However, some studies indicate that the changes in F-actin are not a key to changes in ion transport but, rather, are a direct result of the alterations in ion flux or content. Finally, not all cell types respond the same way to cytoskeletal perturbation, indicating that a role for the cytoskeleton may be cell specific rather than fundamental.
STUDY OF SOME M E C H A N I S M S CONTROLLING THE BETAINE UPTAKE CAPACITY OF THE G R A M POSITIVE BACTERIA L A C T O C O C C U S LACTIS NCDO763 Obis D., Guillot A. and Mistou M-Y. Unit6 de Recherches Biochimie et Structure des Prot6ines, INRA, 78352 Jouy-en-Josas cedex, France In L. lactis NCDO763, the activity of an unique ABC transport system called BusA or OpuA is responsible for osmodependent betaine accumulation.~'~'3 BusA is a close relative of OpuA, its functional homolog in B. subtilis, 4 but the lactococcal transport system presents an original fusion event between the membrane and the substrate- binding domains. As in E. coli or B. subtilis, osmotic upshock increases the betaine uptake capacity of L. lactis by raising the transcription of busA and the activity of BusA. Our present work focuses on the mechanisms controlling the osmotic induction of busA. The transcriptional start sites of busA were mapped and two promoters were found to control the expression of busA The distal promoter (PI) presents a consensus -35 region and an extented -10 box : it is silent at low osmolarity and activated above 0.4 osM. A second, proximal promoter (P2) seems to be responsible of a low, basal level of transcription indifferent to the medium osmolarity. The level of luciferase activity in strains expressing chromosomal busA:luxAluxB fusions were measured during growth under osmotic constrainst to specify the regulatory regions necessary for the osmotic induction. These constructs were also used to study the role of compatible solute accumulation on the down-regulation of busA transcription. In L. lactis NCDO763, a gene located upstream of busA codes for a transcriptional regulator of the GntR family. In vivo experiments indicated that this protein acts as a repressor of busA expression. I. 2. 3. 4.
Obis, D. et al. (1999) J. Bacteriol. 181 6238-6246 van der Heide, T. and B. Poolman (2000) J. Bacteriol. 182 203-206 Bouvier, J. et al. (2000) J. Mol. Microbiol. Biotechnol. 2 199-205 Kempf, B. and E. Bremer (1995) J. Biol Chem. 270 16701-13