Synaptic membrane proteins form stable microdomains in early endosomes.
In the plasma membrane, membrane proteins are frequently organized in microdomains that are stabilized both by protein-protein and protein-lipid interactions, with the membrane lipid cholesterol being instrumental for microdomain stability. However, it is unclear whether such microdomains persist during endocytotic membrane trafficking. We used stimulated emission-depletion microscopy to investigate the domain structure of the endosomes. We developed a semiautomatic method for counting the individual domains, an approach that we have validated by immunoelectron microscopy. We found that in endosomes derived from neuroendocrine PC12 cells synaptophysin and several SNARE proteins are organized in microdomains. Cholesterol depletion by methyl-beta-cyclodextrin disintegrates most of the domains. Interestingly, no change in the frequency of microdomains was observed when endosomes were fused with protein-free liposomes of similar size (in what constitutes a novel approach in modifying acutely the lipid composition of organelles), regardless of whether the membrane lipid composition of the liposomes was similar or very different from that of the endosomes. Similarly, Rab depletion from the endosome membranes left the domain structure unaffected. Furthermore, labeled exogenous protein, introduced into endosomes by liposome fusion, equilibrated with the corresponding microdomains. We conclude that synaptic membrane proteins are organized in stable but dynamic clusters within endosomes, which are likely to persist during membrane recycling.
Geumann
Ulf
U
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany.
Schäfer
Christina
C
Riedel
Dietmar
D
Jahn
Reinhard
R
Rizzoli
Silvio O
SO
D000818:Animals; D001923:Brain Chemistry; D002462:Cell Membrane / Q000378:metabolism*; D011992:Endosomes / Q000737:chemistry*; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis*; D008853:Microscopy / Q000379:methods; D051381:Rats; D050600:SNARE Proteins / Q000032:analysis; D013570:Synaptic Membranes / Q000737:chemistry*; D016708:Synaptophysin / Q000032:analysis
D008565:Membrane Proteins; D050600:SNARE Proteins; D016708:Synaptophysin