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1.
J Cell Biol ; 211(1): 123-44, 2015 Oct 12.
Article in English | MEDLINE | ID: mdl-26459602

ABSTRACT

Endocytosis allows cargo to enter a series of specialized endosomal compartments, beginning with early endosomes harboring Rab5 and its effector EEA1. There are, however, additional structures labeled by the Rab5 effector APPL1 whose role in endocytic transport remains unclear. It has been proposed that APPL1 vesicles are transport intermediates that convert into EEA1 endosomes. Here, we tested this model by analyzing the ultrastructural morphology, kinetics of cargo transport, and stability of the APPL1 compartment over time. We found that APPL1 resides on a tubulo-vesicular compartment that is capable of sorting cargo for recycling or degradation and that displays long lifetimes, all features typical of early endosomes. Fitting mathematical models to experimental data rules out maturation of APPL1 vesicles into EEA1 endosomes as a primary mechanism for cargo transport. Our data suggest instead that APPL1 endosomes represent a distinct population of Rab5-positive sorting endosomes, thus providing important insights into the compartmental organization of the early endocytic pathway.


Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Endocytosis , Endosomes/metabolism , HeLa Cells , Humans , Protein Transport
2.
Traffic ; 16(1): 68-84, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25367362

ABSTRACT

Retromer, a peripheral membrane protein complex, plays an instrumental role in host of cellular processes by its ability to recycle receptors from endosomes to the trans-Golgi network. It consists of two distinct sub-complexes, a membrane recognizing, sorting nexins (SNX) complex and a cargo recognition, vacuolar protein sorting (Vps) complex. Small GTPase, Rab7 is known to recruit retromer on endosomal membrane via interactions with the Vps sub-complex. The molecular mechanism underlying the recruitment process including the role of individual Vps proteins is yet to be deciphered. In this study, we developed a FRET-based assay in HeLa cells that demonstrated the interaction of Rab7 with Vps35 and Vps26 in vivo. Furthermore, we showed that Rab7 recruits retromer to late endosomes via direct interactions with N-terminal conserved regions in Vps35. However, the single point mutation, which disrupts the interaction between Vps35 and Vps26, perturbed the Rab7-mediated recruitment of retromer in HeLa cells. Using biophysical measurements, we demonstrate that the association of Vps26 with Vps35 resulted in high affinity binding between the Vps sub-complex and the activated Rab7 suggesting for a possible allosteric role of Vps26. Thus, this study provides molecular insights into the essential role of Vps26 and Vps35 in Rab7-mediated recruitment of the core retromer complex.


Subject(s)
Endosomes/metabolism , Intracellular Membranes/metabolism , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/metabolism , trans-Golgi Network/metabolism , HeLa Cells , Humans , rab7 GTP-Binding Proteins
3.
Cell Host Microbe ; 13(2): 129-42, 2013 Feb 13.
Article in English | MEDLINE | ID: mdl-23414754

ABSTRACT

Pharmacological modulators of host-microbial interactions can in principle be identified using high-content screens. However, a severe limitation of this approach is the lack of insights into the mode of action of compounds selected during the primary screen. To overcome this problem, we developed a combined experimental and computational approach. We designed a quantitative multiparametric image-based assay to measure intracellular mycobacteria in primary human macrophages, screened a chemical library containing FDA-approved drugs, and validated three compounds for intracellular killing of M. tuberculosis. By integrating the multiparametric profiles of the chemicals with those of siRNAs from a genome-wide survey on endocytosis, we predicted and experimentally verified that two compounds modulate autophagy, whereas the third accelerates endosomal progression. Our findings demonstrate the value of integrating small molecules and genetic screens for identifying cellular mechanisms modulated by chemicals. Furthermore, selective pharmacological modulation of host trafficking pathways can be applied to intracellular pathogens beyond mycobacteria.


Subject(s)
Anti-Bacterial Agents/pharmacology , Autophagy/drug effects , Mycobacterium tuberculosis/drug effects , RNA Interference , Anti-Bacterial Agents/chemistry , Biological Transport , Colony Count, Microbial , Computational Biology/methods , Endocytosis , Endosomes , Green Fluorescent Proteins/metabolism , Haloperidol/chemistry , Haloperidol/pharmacology , HeLa Cells , Host-Pathogen Interactions/drug effects , Humans , Macrophages/drug effects , Macrophages/microbiology , Macrophages/ultrastructure , Microbial Sensitivity Tests , Microbial Viability/drug effects , Mycobacterium tuberculosis/pathogenicity , Nortriptyline/chemistry , Nortriptyline/pharmacology , Phagosomes , Prochlorperazine/chemistry , Prochlorperazine/pharmacology
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