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Ultrafast diffusion of a fluorescent cholesterol analog in compartmentalized plasma membranes.

ABSTRACT: Cholesterol distribution and dynamics in the plasma membrane (PM) are poorly understood. The recent development of Bodipy488-conjugated cholesterol molecule (Bdp-Chol) allowed us to study cholesterol behavior in the PM, using single fluorescent-molecule imaging. Surprisingly, in the intact PM, Bdp-Chol diffused at the fastest rate ever found for any molecules in the PM, with a median diffusion coefficient (D) of 3.4 µm²/second, which was ?10 times greater than that of non-raft phospholipid molecules (0.33?µm²/second), despite Bdp-Chol's probable association with raft domains. Furthermore, Bdp-Chol exhibited no sign of entrapment in time scales longer than 0.5?milliseconds. In the blebbed PM, where actin filaments were largely depleted, Bdp-Chol and Cy3-conjugated dioleoylphosphatidylethanolamine (Cy3-DOPE) diffused at comparable Ds (medians?=?5.8 and 6.2 µm²/second, respectively), indicating that the actin-based membrane skeleton reduces the D of Bdp-Chol only by a factor of ?2 from that in the blebbed PM, whereas it reduces the D of Cy3-DOPE by a factor of ?20. These results are consistent with the previously proposed model, in which the PM is compartmentalized by the actin-based membrane-skeleton fence and its associated transmembrane picket proteins for the macroscopic diffusion of all of the membrane molecules, and suggest that the probability of Bdp-Chol passing through the compartment boundaries, once it enters the boundary, is ?10× greater than that of Cy3-DOPE. Since the compartment sizes are greater than those of the putative raft domains, we conclude that raft domains coexist with membrane-skeleton-induced compartments and are contained within them.

SUBMITTER: Hiramoto-Yamaki N

PROVIDER: S-EPMC4265843 | biostudies-other | 2014 Jun

REPOSITORIES: biostudies-other

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Ultrafast diffusion of a fluorescent cholesterol analog in compartmentalized plasma membranes.

Hiramoto-Yamaki Nao N Tanaka Kenji A K KA Suzuki Kenichi G N KG Hirosawa Koichiro M KM Miyahara Manami S H MS Kalay Ziya Z Tanaka Koichiro K Kasai Rinshi S RS Kusumi Akihiro A Fujiwara Takahiro K TK

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Cholesterol distribution and dynamics in the plasma membrane (PM) are poorly understood. The recent development of Bodipy488-conjugated cholesterol molecule (Bdp-Chol) allowed us to study cholesterol behavior in the PM, using single fluorescent-molecule imaging. Surprisingly, in the intact PM, Bdp-Chol diffused at the fastest rate ever found for any molecules in the PM, with a median diffusion coefficient (D) of 3.4 µm²/second, which was ∼10 times greater than that of non-raft phospholipid molec ...[more]

PMID: 24506328

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Project description:A uniquely sensitive method for ceramide domain detection allowed us to study in detail cholesterol-ceramide interactions in lipid bilayers with low (physiological) ceramide concentrations, ranging from low or no cholesterol (a situation similar to intracellular membranes, such as endoplasmic reticulum) to high cholesterol (similar to mammalian plasma membrane). Diverse fluorescence spectroscopy and microscopy experiments were conducted showing that for low cholesterol amounts ceramide segregates into gel domains that disappear upon increasing cholesterol levels. This was observed in different raft (sphingomyelin/cholesterol-containing) and non-raft (sphingomyelin-absent) membranes, i.e. mimicking different types of cell membranes. Cholesterol-ceramide interactions have been described mainly as raft sphingomyelin-dependent. Here sphingomyelin independence is demonstrated. In addition, ceramide-rich domains re-appear when either cholesterol is converted by cholesterol oxidase to cholestenone or the temperature is decreased. Ceramide is more soluble in cholesterol-rich fluid membranes than in cholesterol-poor ones, thereby increasing the chemical potential of cholesterol. Ceramide solubility depends on the average gel-fluid transition temperature of the remaining membrane lipids. The inability of cholestenone-rich membranes to dissolve ceramide gel domains shows that the cholesterol ordering and packing properties are fundamental to the mixing process. We also show that the solubility of cholesterol in ceramide domains is low. The results are rationalized by a ternary phospholipid/ceramide/cholesterol phase diagram, providing the framework for the better understanding of biochemical phenomena modulated by cholesterol-ceramide interactions such as cholesterol oxidase activity, lipoprotein metabolism, and lipid targeting in cancer therapy. It also suggests that the lipid compositions of different organelles are such that ceramide gel domains are not formed unless a stress or pathological situation occurs.

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Ultrafast diffusion of a fluorescent cholesterol analog in compartmentalized plasma membranes.

Publications

Ultrafast diffusion of a fluorescent cholesterol analog in compartmentalized plasma membranes.

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