Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization

Abstract

The formation and maintenance of polarized distributions of membrane proteins in the cell membrane are key to the function of polarized cells. In polarized neurons, various membrane proteins are localized to the somatodendritic domain or the axon. Neurons control polarized delivery of membrane proteins to each domain, and in addition, they must also block diffusional mixing of proteins between these domains. However, the presence of a diffusion barrier in the cell membrane of the axonal initial segment (IS), which separates these two domains, has been controversial: it is difficult to conceive barrier mechanisms by which an even diffusion of phospholipids could be blocked. Here, by observing the dynamics of individual phospholipid molecules in the plasma membrane of developing hippocampal neurons in culture, we found that their diffusion was blocked in the IS membrane. We also found that the diffusion barrier is formed in neurons 7-10 days after birth through the accumulation of various transmembrane proteins that are anchored to the dense actin-based membrane skeleton meshes being formed under the IS membrane. We conclude that various membrane proteins anchored to the dense membrane skeleton function as rows of pickets, which even stop the overall diffusion of phospholipids, and may represent a universal mechanism for formation of diffusion barriers in the cell membrane.

Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories

“…The observed elongated clusters and irregular domain boundaries suggest that line tension (and thus lipid fluid phase behavior) plays a limited role in domain shape (24), but interactions between the cytoskeleton and membrane proteins could certainly produce such a distribution of HA clusters within which lipids and proteins are confined but diffuse locally, such as has been suggested by Edidin, Kusumi, Vale, and others (16,17,26,27). Interdependence of raft protein trafficking, lipid second messengers, and the cytoskeleton merit further investigation (27)(28)(29).…”

“…However, consensus on the existence, structure, size, and dynamics of rafts continues to be elusive because of disparate results obtained by a variety of methods (13). Instead, rafts are defined principally by biochemical means (14,15), and numerous biophysical models including lipid shells (5), temporary lateral confinement (16,17), preferential partitioning (18), and nanodomains (15,19) cannot be distinguished easily because they predict crucial structural details on length scales much smaller than the wavelength of visible light. Further distinction between these membrane models would provide insight into a large number of biological processes that depend on rafts (5,15,20).…”

Dynamics of bacteriorhodopsin 2D crystal observed by high-speed atomic force microscopy

“…In particular, it has been shown that proteins in native membranes may not diffuse freely but are in fact confined to specific domains. Cells use several confining mechanisms such as anchoring to the cytoskeleton through hetero-bifunctional proteins (Byers and Branton, 1985), diffusion barriers formed by the accumulation of proteins anchored to cytoskeleton meshes (Halenda et al, 1987;Nakada et al, 2003), or self-assembly into large two-dimensional (2D) crystalline patches, as in the case of bacteriorhodopsin (bR), a light-driven proton pump found in the so-called purple membranes (PM) of Halobacterium salinarum (Blaurock, 1971). Despite these advances, an understanding of the membrane dynamics at the nanoscale remains a major challenge due primarily to the lack of measurement techniques allowing simultaneous spatial and temporal observation of single molecules within native membranes.…”

Transmembrane Dynamics of Lipids

“…It is also addressed to physicists fascinated by the various spontaneous self -organization of lipids in water (lipid polymorphism) to warn them that lipids in biological systems are not always at thermal equilibrium, and that phase separations and lateral or transmembrane domains seen in model systems can differ fundamentally from biological situations. Indeed, molecule segregation in biological systems results often from the work of ATPases, like the fl ippases, or is the result of a molecule sorting by " protein gates " (see the " fence and picket model " of Kusumi and collaborators [2] ). Such mechanisms are diffi cult to mimic in model systems.…”

“…Why are there so many chemically different lipids coexisting in nature? (2) Why is the lipid composition of various membranes of eukaryotes different and sometimes even the two sides of biological membranes different (asymmetrical)? This requires numerous specifi c enzymes for the synthesis and ultimately for the shuttling to the right destination of newly formed lipids.…”