Dataset Information

Tracking single serotonin transporter molecules at the endoplasmic reticulum and plasma membrane.

ABSTRACT: Transmembrane proteins are synthesized and folded in the endoplasmic reticulum (ER), an interconnected network of flattened sacs or tubes. Up to now, this organelle has eluded a detailed analysis of the dynamics of its constituents, mainly due to the complex three-dimensional morphology within the cellular cytosol, which precluded high-resolution, single-molecule microscopy approaches. Recent evidences, however, pointed out that there are multiple interaction sites between ER and the plasma membrane, rendering total internal reflection microscopy of plasma membrane proximal ER regions feasible. Here we used single-molecule fluorescence microscopy to study the diffusion of the human serotonin transporter at the ER and the plasma membrane. We exploited the single-molecule trajectories to map out the structure of the ER close to the plasma membrane at subdiffractive resolution. Furthermore, our study provides a comparative picture of the diffusional behavior in both environments. Under unperturbed conditions, the majority of proteins showed similar mobility in the two compartments; at the ER, however, we found an additional 15% fraction of molecules moving with 25-fold faster mobility. Upon degradation of the actin skeleton, the diffusional behavior in the plasma membrane was strongly influenced, whereas it remained unchanged in the ER.

SUBMITTER: Anderluh A

PROVIDER: S-EPMC4017317 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Tracking single serotonin transporter molecules at the endoplasmic reticulum and plasma membrane.

Anderluh Andreas A Klotzsch Enrico E Ries Jonas J Reismann Alexander W A F AW Weber Stefan S Fölser Martin M Koban Florian F Freissmuth Michael M Sitte Harald H HH Schütz Gerhard J GJ

Biophysical journal 20140501 9

Transmembrane proteins are synthesized and folded in the endoplasmic reticulum (ER), an interconnected network of flattened sacs or tubes. Up to now, this organelle has eluded a detailed analysis of the dynamics of its constituents, mainly due to the complex three-dimensional morphology within the cellular cytosol, which precluded high-resolution, single-molecule microscopy approaches. Recent evidences, however, pointed out that there are multiple interaction sites between ER and the plasma memb ...[more]

PMID: 24806941

Similar Datasets

Project description:Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

Dataset Information

Tracking single serotonin transporter molecules at the endoplasmic reticulum and plasma membrane.

Publications

Tracking single serotonin transporter molecules at the endoplasmic reticulum and plasma membrane.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets