endocytic compartment
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2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mai Anh Do ◽  
Daniel Levy ◽  
Annie Brown ◽  
Gerard Marriott ◽  
Biao Lu

AbstractTargeted delivery of lysosomal enzymes to the endocytic compartment of human cells represents a transformative technology for treating a large family of lysosomal storage diseases (LSDs). Gaucher disease is one of the most common types of LSDs caused by mutations to the lysosomal β-glucocerebrosidase (GBA). Here, we describe a genetic strategy to produce engineered exosomes loaded with GBA in two different spatial configurations for targeted delivery to the endocytic compartment of recipient cells. By fusing human GBA to an exosome-anchoring protein: vesicular stomatitis virus glycoprotein (VSVG), we demonstrate that the chimeric proteins were successfully integrated into exosomes which were secreted as extracellular vesicles (EVs) by producer cells. Isolation and molecular characterization of EVs confirmed that the fusion proteins were loaded onto exosomes without altering their surface markers, particle size or distribution. Further, enzyme-loaded exosomes/EVs added to cultured medium were taken up by recipient cells. Further, the endocytosed exosomes/EVs targeted to endocytic compartments exhibited a significant increase in GBA activity. Together, we have developed a novel method for targeting and delivery of lysosomal enzymes to their natural location: the endocytic compartment of recipient cells. Since exosomes/EVs have an intrinsic ability to cross the blood-brain-barrier, our technology may provide a new approach to treat severe types of LSDs, including Gaucher disease with neurological complications.


2016 ◽  
Vol 182 ◽  
pp. 11-21
Author(s):  
Mette Schjaerff ◽  
Stefan M. Keller ◽  
Verena K. Affolter ◽  
Annemarie T. Kristensen ◽  
Peter F. Moore

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Junko Y Toshima ◽  
Eri Furuya ◽  
Makoto Nagano ◽  
Chisa Kanno ◽  
Yuta Sakamoto ◽  
...  

The actin cytoskeleton plays important roles in the formation and internalization of endocytic vesicles. In yeast, endocytic vesicles move towards early endosomes along actin cables, however, the molecular machinery regulating interaction between endocytic vesicles and actin cables is poorly understood. The Eps15-like protein Pan1p plays a key role in actin-mediated endocytosis and is negatively regulated by Ark1 and Prk1 kinases. Here we show that pan1 mutated to prevent phosphorylation at all 18 threonines, pan1-18TA, displayed almost the same endocytic defect as ark1Δ prk1Δ cells, and contained abnormal actin concentrations including several endocytic compartments. Early endosomes were highly localized in the actin concentrations and displayed movement along actin cables. The dephosphorylated form of Pan1p also caused stable associations between endocytic vesicles and actin cables, and between endocytic vesicles and endosomes. Thus Pan1 phosphorylation is part of a novel mechanism that regulates endocytic compartment interactions with each other and with actin cables.


2012 ◽  
Vol 86 (16) ◽  
pp. 8346-8358 ◽  
Author(s):  
Bernardo A. Mainou ◽  
Terence S. Dermody

Rab GTPases play an essential role in vesicular transport by coordinating the movement of various types of cargo from one cellular compartment to another. Individual Rab GTPases are distributed to specific organelles and thus serve as markers for discrete types of endocytic vesicles. Mammalian reovirus binds to cell surface glycans and junctional adhesion molecule-A (JAM-A) and enters cells by receptor-mediated endocytosis in a process dependent on β1 integrin. Within organelles of the endocytic compartment, reovirus undergoes stepwise disassembly catalyzed by cathepsin proteases, which allows the disassembly intermediate to penetrate endosomal membranes and release the transcriptionally active viral core into the cytoplasm. The pathway used by reovirus to traverse the endocytic compartment is largely unknown. In this study, we found that reovirus particles traffic through early, late, and recycling endosomes during cell entry. After attachment to the cell surface, reovirus particles and JAM-A codistribute into each of these compartments. Transfection of cells with constitutively active and dominant-negative Rab GTPases that affect early and late endosome biogenesis and maturation influenced reovirus infectivity. In contrast, reovirus infectivity was not altered in cells expressing mutant Rab GTPases that affect recycling endosomes. Thus, reovirus virions localize to early, late, and recycling endosomes during entry into host cells, but only those that traverse early and late endosomes yield a productive infection.


2012 ◽  
Vol 13 (1) ◽  
pp. 29 ◽  
Author(s):  
Elizabeth F Noratel ◽  
Chere’ L Petty ◽  
Jessica S Kelsey ◽  
Hoa N Cost ◽  
Nisha Basappa ◽  
...  

2009 ◽  
Vol 335 (1-2) ◽  
pp. 273-282 ◽  
Author(s):  
Brunella Tancini ◽  
Alessandro Magini ◽  
Loredana Latterini ◽  
Lorena Urbanelli ◽  
Virginia Ciccarone ◽  
...  

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