The endocytic recycling compartment maintains cargo segregation acquired upon exit from the sorting endosome.
The endocytic recycling compartment (ERC) is a series of perinuclear tubular and vesicular membranes that regulates recycling to the plasma membrane. Despite evidence that cargo is sorted at the early/sorting endosome (SE), whether cargo mixes downstream at the ERC or remains segregated is an unanswered question. Here we use three-dimensional (3D) structured illumination microscopy and dual-channel and 3D direct stochastic optical reconstruction microscopy (dSTORM) to obtain new information about ERC morphology and cargo segregation. We show that cargo internalized either via clathrin-mediated endocytosis (CME) or independently of clathrin (CIE) remains segregated in the ERC, likely on distinct carriers. This suggests that no further sorting occurs upon cargo exit from SE. Moreover, 3D dSTORM data support a model in which some but not all ERC vesicles are tethered by contiguous "membrane bridges." Furthermore, tubular recycling endosomes preferentially traffic CIE cargo and may originate from SE membranes. These findings support a significantly altered model for endocytic recycling in mammalian cells in which sorting occurs in peripheral endosomes and segregation is maintained at the ERC.
Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-5870.
Shuwei
S
Xie
Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-5870.
Kriti
K
Bahl
Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-5870.
James B
JB
Reinecke
Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
Gerald R V
GR
Hammond
Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-5870.
Naava
N
Naslavsky
Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-5870 scaplan@unmc.edu.
Steve
S
Caplan
D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008853:Microscopy / Q000379:methods; D021381:Protein Transport / Q000502:physiology; D014168:Transferrin / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism*
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D014168:Transferrin