In this study, we show that cell surface area of oligodendrocytes is critically dependent on actomyosin contractility and is regulated by the physical properties of the supporting matrix. Furthermore, we find that chondroitin sulfate proteoglycans (CSPG), molecules associated with non-permissive growth properties within the central nervous system (CNS), block cell surface spreading. In addition, we observed that the reductions of plasma membrane area were accompanied by changes in the rate of fluid-phase endocytosis. We found that this form of endocytosis is dependent on actomyosin contractility and possibly also on integrins. A possible explanation is that actomyosin contractility pulls membrane into the cell and thereby accelerates vesicle budding. Loss of integrin-mediated anchoring to the extracellular matrix may increase these pulling forces of the actin cytoskeleton. We show that this mechanism is important for cell surface spreading of Oli-neu cells, but it is likely to be involved in the control of cell surface area in other systems. It may be particularly relevant in processes where rapid changes of cell surface area occur, for example in a blood vessel or a bladder [27
Previous work shows that extracellular matrix proteins, like laminin, promote the formation of myelin membrane sheets in oligodendrocytes by its interaction with integrin α6β1 [28
]. Oligodendrocytes are known to express a large number of additional integrins including αvβ1, αvβ3 and α5β5, which may regulate different aspects of oligodendrocyte biology such as proliferation, differentiation and survival [9
In addition to signals from the extracellular matrix, paracrine signals from oligodendrocytes may also regulate myelin-membrane extension. Interestingly, the effects of the CSPG peptide on oligodendrocyte cell surface area was not observed when cells were grown at a high density, indicating oligodendrocyte present factors to neighboring cells that are able to overcome the inhibitory cues.
Integrin-mediated cell attachment is known to regulate endocytosis and cell surface shape. A striking example of the importance of this mechanism is the loss of integrin-mediated adhesion during tumor growth, which triggers rapid endocytosis and cell rounding [1
]. The growth promoting properties of extracellular matrix proteins in oligodendrocytes might thus in part be explained by integrin-mediated retention of membrane at the cell surface. In addition, a role of extracellular matrix proteins in regulating vesicular traffic within the biosynthetic pathway to the myelin membrane sheets has been proposed [35
The finding that actomyosin contractility acts downstream of integrins is consistent with a report describing a signal transduction pathway from integrins to Fyn that facilitates RhoA GTPase inactivation and the morphological differentiation of oligodendrocytes [10
]. Our results extend this pathway by providing evidence that myosin II is a downstream target that is regulated by integrins and Rho/ROCK activity. This concurs with the finding that myosin II inactivation promotes myelination in the CNS [36
In the adult brain OPCs are kept in an undifferentiated state. This may in part be due to the presence of myelin from mature oligodendrocytes that blocks their differentiation [37
]. Whether this occurs by Rho/ROCK/myosin II activation is an interesting question for future research. After demyelination OPCs are recruited to the site of the lesion where they differentiate. In multiple sclerosis remyelination can be extensive, however, OPC frequently fail to differentiate [38
]. The inhibitory cues for OPCs in multiple sclerosis lesions have not been identified, but upregulation of growth inhibiting molecules such as CSPG within the glial scar is one possibility.
We do not know by what means CSPGs affects cell surface size, but the finding that knocking down myosin II overcomes CSPGs inhibitory effects points to a role of actomyosin contractility in this process. Earlier work examining the role of CSPGs on axon outgrowth suggested that CSPGs interfere with extracellular matrix/integrin interactions [42
Indeed, increased integrin expression is sufficient to eliminate the inhibitory effects of CSPGs on neuronal growth cone function [43
]. A down-stream role of the Rho/ROCK signalling system has also been identified [25
Our results demonstrating that inhibition of Rho/ROCK/myosin II activity is able to overcome a non-permissive growth property of the extracellular environment on oligodendrocytes extends the role of this system in cellular regeneration.
These findings not only show that the actomyosin system plays a role in cell surface spreading of oligodendrocytes, but also point to potential target for therapeutic interventions. However, we do not yet know whether the inhibition of actomyosin contractility affects the ability of oligodendrocytes to wrap their plasma membrane around an axon to form compact myelin. Clearly, in vivo studies are now required to determine whether inhibition of actomyosin contractility promotes myelin repair in the CNS.