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Interplay between Endothelial Cell Cytoskeletal Rigidity and Plasma Membrane Fluidity
New and Notable
Interplay between Endothelial Cell Cytoskeletal Rigidity and Plasma Membrane Fluidity Gary J. Blanchard1,* and Julia V. Busik2 1
Department of Chemistry, and 2Department of Physiology, Michigan State University, East Lansing, Michigan
The biomechanical properties of endothelial cells are critically important for multiple endothelial cell functions including permeability, extravasation of immune cells, migration, and angiogenesis. From a biomechanical standpoint, the two primary parameters are the deformability of the cell and the fluid properties of its plasma membrane. (The terms ‘‘deformability’’ and ‘‘fluid properties’’ are used extensively in the literature and, as applied to plasma membranes, they have specific meanings. The term ‘‘deformability’’ is typically taken to mean ‘‘elastic modulus’’, a property that is measured using AFM, with the modulus being extracted from the data using the Hertz model. The term ‘‘fluid properties’’ or ‘‘fluidity’’ is typically taken to mean the viscosity of the plasma membrane, a property determined from the diffusion constant (either rotational or translational) of a membrane constituent.) These two parameters, which are determined by two different cellular structures, the F-actin network and the plasma membrane, cannot be controlled independently because of the complex and still evolving understanding of the interactions between them. While it may be tempting to start with the assumption that the plasma
Submitted December 30, 2016, and accepted for publication January 23, 2017. *Correspondence: [email protected] Editor: Claudia Steinem.
membrane simply rides on the cytoskeletal structure, this is not the case, and Levitan and co-workers (1–3) have pioneered the effort to elucidate the subtle and complex molecular interactions responsible for this codependency. The two properties of cell deformability and membrane fluidity play complementary roles in immune cell extravasation and other permeabilityrelated functions in endothelial cells, as is schematized in Fig. 1. While gaining systematic structural control over cytoskeletal rigidity poses a substantial challenge, the plasma membrane, in contrast, is amenable to facile changes in composition, either by exposure to constituents or by the action of enzymes (e.g., acid sphingomyelinase conversion of sphingomyelin to ceramide) (4,5), or by oxidation of selected species, as is considered in the latest work from the Levitan group (Ayee et al. (6)). Indeed, exploring the ability to control interactions between the plasma membrane and the cytoskeletal structure through the composition of the plasma membrane is at once a promising avenue and an exceptionally complex effort owing to the compositional heterogeneity of the membrane and the limited extent to which the interactions between these two cellular entities are understood. It is well established that the composition of the plasma membrane affects its ‘‘rigidity’’, which is taken to mean
its fluidity. It is perhaps the use of the term ‘‘rigidity’’ in this context that has made the distinction between the role of the plasma membrane and the cytoskeleton less clear than it should be. Cholesterol, for example, is widely understood to play a role in determining the fluidity of the plasma membrane, with the addition of cholesterol being said to stiffen the structure (7). Levitan and co-workers (2,3) have demonstrated previously that the amount of cholesterol in the plasma membrane of endothelial cells also influences the extent to which the plasma membrane interacts with the cytoskeleton. This interesting result naturally drives the questions of 1) what other species in the plasma membrane interact with the cytoskeleton? and, with this knowledge, 2) what is the molecular nature of these interactions? Recognizing that oxidation is a key biological process that can alter the composition of the plasma membrane, the Levitan group (Ayee et al. (6)) have focused on the use of oxidation products of selected phospholipids to evaluate not only the chemical structure dependence of the interactions between the oxidation products and the plasma membrane, but also how such changes alter the membrane interactions with the cytoskeletal structure. The Levitan group (Ayee et al. (6)) focuses on the phosphocholine 1-palmitoyl-2-arachidonoyl-sn-glycerophosphocholine (PAPC) because the
http://dx.doi.org/10.1016/j.bpj.2017.01.013 Ó 2017 Biophysical Society.
Biophysical Journal 112, 831–833, March 14, 2017 831
Blanchard and Busik
membrane fluidity plasma membrane points of interaction
pressure
endothelial cell
vascular smooth muscle cells FIGURE 1 Schematic of interplay between endothelial cell deformability and plasma membrane fluidity. To see this figure in color, go online.
unsaturations in this asymmetric phospholipid are capable of undergoing oxidative degradation, and the products affect the properties of the plasma membrane. In particular, two of the oxidation products, 1-palmitoyl-2(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC, an aldehyde) and 1-palmitoyl-2-glytaroyl-sn-glycero-3phosphocholine (PGPC, a carboxylic acid) (Fig. 2) have been examined
both experimentally and theoretically to understand the effects of these structural modifications on the organization of the plasma membrane and on the rigidity of the cell. The authors find, essentially, that the polarity of the terminal oxidized moieties of POVPC and PGPC interact preferentially with regions of different polarity in the plasma membrane, owing to the different dipole moments of the
1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine (PAPC)
1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC)
oxidized terminal groups and the ability of the carboxylic acid (PGPC) to undergo hydrogen bonding and dissociation. By combining sophisticated spectroscopic methods with molecular dynamics simulations, the authors are able to gauge not only the extent to which the plasma membrane organization is changed, but also can infer the extent to which these changes alter the rigidity of the endothelial cell. This work establishes in a compelling manner that a variety of plasma membrane constituents—not just cholesterol—are involved in both the morphology and fluidity of the plasma membrane as well as the rigidity of the endothelial cell. There is thus a range of molecular-scale interactions responsible for the relationship between plasma membrane fluidity and cellular rigidity, with the extent and nature of those interactions waiting to be elucidated fully. This nicely executed study leaves several questions open for further investigation. First, it will be important to determine whether the same rules apply to the cells with less developed actin networks, such as immune cells and progenitor cells. In addition, HUVECs were used as a model system in the study. As it is appreciated that microvascular and macrovascular, as well as venous and arterial cells, behave very differently in regard to immune cell extravasation, migration, and angiogenesis, it will be important to address the cross talk between membrane lipids and cell deformability in aortic endothelial cells, as well as microvascular endothelial cells from both fenestrated and barrier capillary beds. REFERENCES 1. Byfield, F. J., H. Aranda-Espinoza, ., I. Levitan. 2004. Cholesterol depletion increases membrane stiffness of aortic endothelial cells. Biophys. J. 87:3336–3343.
1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC)
FIGURE 2 Chemical structures of phospholipids (PAPC, POVPC, and PGPC) used in the study by Ayee et al. (6).
832 Biophysical Journal 112, 831–833, March 14, 2017
2. Sun, M., N. Northup, ., G. Forgacs. 2007. The effect of cellular cholesterol on membrane-cytoskeleton adhesion. J. Cell Sci. 120:2223–2231. 3. Ayee, M. A., and I. Levitan. 2016. Paradoxical impact of cholesterol on lipid packing
New and Notable and cell stiffness. Front. Biosci. (Landmark Ed.). 21:1245–1259. 4. Zidovetzki, R., and I. Levitan. 2007. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. Biochim. Biophys. Acta. 1768:1311–1324.
5. Chakravarthy, H., S. Navitskaya, ., J. V. Busik. 2016. Role of acid sphingomyelinase in shifting the balance between proinflammatory and reparative bone marrow cells in diabetic retinopathy. Stem Cells. 34:972–983. 6. Ayee, M. A., E. LeMaster, ., I. Levitan. 2017. Molecular-scale biophysical modula-
tion of an endothelial membrane by oxidized phospholipids. Biophys. J. 112:325–338. 7. Pillman, H. A., and G. J. Blanchard. 2011. Consequences of transient heating on the motional dynamics of cholesterol-containing phospholipid vesicles. J. Phys. Chem. B. 115: 3819–3827.
Biophysical Journal 112, 831–833, March 14, 2017 833
Interplay between Endothelial Cell Cytoskeletal Rigidity and Plasma Membrane Fluidity Gary J. Blanchard1,* and Julia V. Busik2 1
Department of Chemistry, and 2Department of Physiology, Michigan State University, East Lansing, Michigan
The biomechanical properties of endothelial cells are critically important for multiple endothelial cell functions including permeability, extravasation of immune cells, migration, and angiogenesis. From a biomechanical standpoint, the two primary parameters are the deformability of the cell and the fluid properties of its plasma membrane. (The terms ‘‘deformability’’ and ‘‘fluid properties’’ are used extensively in the literature and, as applied to plasma membranes, they have specific meanings. The term ‘‘deformability’’ is typically taken to mean ‘‘elastic modulus’’, a property that is measured using AFM, with the modulus being extracted from the data using the Hertz model. The term ‘‘fluid properties’’ or ‘‘fluidity’’ is typically taken to mean the viscosity of the plasma membrane, a property determined from the diffusion constant (either rotational or translational) of a membrane constituent.) These two parameters, which are determined by two different cellular structures, the F-actin network and the plasma membrane, cannot be controlled independently because of the complex and still evolving understanding of the interactions between them. While it may be tempting to start with the assumption that the plasma
Submitted December 30, 2016, and accepted for publication January 23, 2017. *Correspondence: [email protected] Editor: Claudia Steinem.
membrane simply rides on the cytoskeletal structure, this is not the case, and Levitan and co-workers (1–3) have pioneered the effort to elucidate the subtle and complex molecular interactions responsible for this codependency. The two properties of cell deformability and membrane fluidity play complementary roles in immune cell extravasation and other permeabilityrelated functions in endothelial cells, as is schematized in Fig. 1. While gaining systematic structural control over cytoskeletal rigidity poses a substantial challenge, the plasma membrane, in contrast, is amenable to facile changes in composition, either by exposure to constituents or by the action of enzymes (e.g., acid sphingomyelinase conversion of sphingomyelin to ceramide) (4,5), or by oxidation of selected species, as is considered in the latest work from the Levitan group (Ayee et al. (6)). Indeed, exploring the ability to control interactions between the plasma membrane and the cytoskeletal structure through the composition of the plasma membrane is at once a promising avenue and an exceptionally complex effort owing to the compositional heterogeneity of the membrane and the limited extent to which the interactions between these two cellular entities are understood. It is well established that the composition of the plasma membrane affects its ‘‘rigidity’’, which is taken to mean
its fluidity. It is perhaps the use of the term ‘‘rigidity’’ in this context that has made the distinction between the role of the plasma membrane and the cytoskeleton less clear than it should be. Cholesterol, for example, is widely understood to play a role in determining the fluidity of the plasma membrane, with the addition of cholesterol being said to stiffen the structure (7). Levitan and co-workers (2,3) have demonstrated previously that the amount of cholesterol in the plasma membrane of endothelial cells also influences the extent to which the plasma membrane interacts with the cytoskeleton. This interesting result naturally drives the questions of 1) what other species in the plasma membrane interact with the cytoskeleton? and, with this knowledge, 2) what is the molecular nature of these interactions? Recognizing that oxidation is a key biological process that can alter the composition of the plasma membrane, the Levitan group (Ayee et al. (6)) have focused on the use of oxidation products of selected phospholipids to evaluate not only the chemical structure dependence of the interactions between the oxidation products and the plasma membrane, but also how such changes alter the membrane interactions with the cytoskeletal structure. The Levitan group (Ayee et al. (6)) focuses on the phosphocholine 1-palmitoyl-2-arachidonoyl-sn-glycerophosphocholine (PAPC) because the
http://dx.doi.org/10.1016/j.bpj.2017.01.013 Ó 2017 Biophysical Society.
Biophysical Journal 112, 831–833, March 14, 2017 831
Blanchard and Busik
membrane fluidity plasma membrane points of interaction
pressure
endothelial cell
vascular smooth muscle cells FIGURE 1 Schematic of interplay between endothelial cell deformability and plasma membrane fluidity. To see this figure in color, go online.
unsaturations in this asymmetric phospholipid are capable of undergoing oxidative degradation, and the products affect the properties of the plasma membrane. In particular, two of the oxidation products, 1-palmitoyl-2(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC, an aldehyde) and 1-palmitoyl-2-glytaroyl-sn-glycero-3phosphocholine (PGPC, a carboxylic acid) (Fig. 2) have been examined
both experimentally and theoretically to understand the effects of these structural modifications on the organization of the plasma membrane and on the rigidity of the cell. The authors find, essentially, that the polarity of the terminal oxidized moieties of POVPC and PGPC interact preferentially with regions of different polarity in the plasma membrane, owing to the different dipole moments of the
1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine (PAPC)
1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC)
oxidized terminal groups and the ability of the carboxylic acid (PGPC) to undergo hydrogen bonding and dissociation. By combining sophisticated spectroscopic methods with molecular dynamics simulations, the authors are able to gauge not only the extent to which the plasma membrane organization is changed, but also can infer the extent to which these changes alter the rigidity of the endothelial cell. This work establishes in a compelling manner that a variety of plasma membrane constituents—not just cholesterol—are involved in both the morphology and fluidity of the plasma membrane as well as the rigidity of the endothelial cell. There is thus a range of molecular-scale interactions responsible for the relationship between plasma membrane fluidity and cellular rigidity, with the extent and nature of those interactions waiting to be elucidated fully. This nicely executed study leaves several questions open for further investigation. First, it will be important to determine whether the same rules apply to the cells with less developed actin networks, such as immune cells and progenitor cells. In addition, HUVECs were used as a model system in the study. As it is appreciated that microvascular and macrovascular, as well as venous and arterial cells, behave very differently in regard to immune cell extravasation, migration, and angiogenesis, it will be important to address the cross talk between membrane lipids and cell deformability in aortic endothelial cells, as well as microvascular endothelial cells from both fenestrated and barrier capillary beds. REFERENCES 1. Byfield, F. J., H. Aranda-Espinoza, ., I. Levitan. 2004. Cholesterol depletion increases membrane stiffness of aortic endothelial cells. Biophys. J. 87:3336–3343.
1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC)
FIGURE 2 Chemical structures of phospholipids (PAPC, POVPC, and PGPC) used in the study by Ayee et al. (6).
832 Biophysical Journal 112, 831–833, March 14, 2017
2. Sun, M., N. Northup, ., G. Forgacs. 2007. The effect of cellular cholesterol on membrane-cytoskeleton adhesion. J. Cell Sci. 120:2223–2231. 3. Ayee, M. A., and I. Levitan. 2016. Paradoxical impact of cholesterol on lipid packing
New and Notable and cell stiffness. Front. Biosci. (Landmark Ed.). 21:1245–1259. 4. Zidovetzki, R., and I. Levitan. 2007. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. Biochim. Biophys. Acta. 1768:1311–1324.
5. Chakravarthy, H., S. Navitskaya, ., J. V. Busik. 2016. Role of acid sphingomyelinase in shifting the balance between proinflammatory and reparative bone marrow cells in diabetic retinopathy. Stem Cells. 34:972–983. 6. Ayee, M. A., E. LeMaster, ., I. Levitan. 2017. Molecular-scale biophysical modula-
tion of an endothelial membrane by oxidized phospholipids. Biophys. J. 112:325–338. 7. Pillman, H. A., and G. J. Blanchard. 2011. Consequences of transient heating on the motional dynamics of cholesterol-containing phospholipid vesicles. J. Phys. Chem. B. 115: 3819–3827.
Biophysical Journal 112, 831–833, March 14, 2017 833