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1.
J Cell Sci ; 136(13)2023 07 01.
Article in English | MEDLINE | ID: mdl-37313743

ABSTRACT

The genetic alterations contributing to migration proficiency, a phenotypic hallmark of metastatic cells required for colonizing distant organs, remain poorly defined. Here, we used single-cell magneto-optical capture (scMOCa) to isolate fast cells from heterogeneous human breast cancer cell populations, based on their migratory ability alone. We show that captured fast cell subpopulations retain higher migration speed and focal adhesion dynamics over many generations as a result of a motility-related transcriptomic profile. Upregulated genes in isolated fast cells encoded integrin subunits, proto-cadherins and numerous other genes associated with cell migration. Dysregulation of several of these genes correlates with poor survival outcomes in people with breast cancer, and primary tumors established from fast cells generated a higher number of circulating tumor cells and soft tissue metastases in pre-clinical mouse models. Subpopulations of cells selected for a highly migratory phenotype demonstrated an increased fitness for metastasis.


Subject(s)
Breast Neoplasms , Neoplastic Cells, Circulating , Animals , Mice , Humans , Female , Breast Neoplasms/pathology , Cell Line, Tumor , Neoplastic Cells, Circulating/pathology , Cell Movement/genetics , Cadherins , Neoplasm Metastasis
2.
Methods Mol Biol ; 2614: 357-368, 2023.
Article in English | MEDLINE | ID: mdl-36587135

ABSTRACT

Single-cell technologies have become critical tools to understand and characterize the complex dynamics that govern biological systems, from embryonic development to cancer heterogeneity. In this context, identification and capture of live individual cells in heterogenous ensembles typically rely on genetic manipulations that encode fluorescent probes. However, a precise understanding of how several molecular components interact to yield the phenotype of interest is a prerequisite to distinguishing and isolating such target cells based on fluorescence alone. Indeed, cellular phenotypes associated with migration, shape, location, or intracellular protein distribution play critical and well-understood roles in cancer biology, but the technologies to tag and isolate cells based on information obtained from imaging are not readily available.Cell labeling via photobleaching (CLaP) and single-cell magneto-optical capture (scMOCa) represent convenient and cost-effective systems for labeling, capturing, and expanding single cells from a heterogenous population, without altering cellular physiology and therefore enabling not only transcriptomic profiling but also biological characterization of target cells. Both techniques allow capturing cells after observation and permit researchers to choose target cells based on information obtained from images. The implementation of these technologies only needs the lasers of a confocal microscope and low-cost, commercially available chemical reagents. Here, we describe a detailed protocol to set up and perform CLaP and scMOCa and highlight critical points for optimal performance.


Subject(s)
Fluorescent Dyes , Light , Fluorescent Dyes/chemistry , Photobleaching , Lasers
3.
Biochem J ; 478(12): 2339-2357, 2021 06 25.
Article in English | MEDLINE | ID: mdl-34060589

ABSTRACT

CLN5 is a soluble endolysosomal protein whose function is poorly understood. Mutations in this protein cause a rare neurodegenerative disease, neuronal ceroid lipofuscinosis (NCL). We previously found that depletion of CLN5 leads to dysfunctional retromer, resulting in the degradation of the lysosomal sorting receptor, sortilin. However, how a soluble lysosomal protein can modulate the function of a cytosolic protein, retromer, is not known. In this work, we show that deletion of CLN5 not only results in retromer dysfunction, but also in impaired endolysosome fusion events. This results in delayed degradation of endocytic proteins and in defective autophagy. CLN5 modulates these various pathways by regulating downstream interactions between CLN3, an endolysosomal integral membrane protein whose mutations also result in NCL, RAB7A, and a subset of RAB7A effectors. Our data support a model where CLN3 and CLN5 function as an endolysosomal complex regulating various functions.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Endosomes/metabolism , Lysosomal Membrane Proteins/metabolism , Lysosomes/metabolism , Membrane Glycoproteins/metabolism , Molecular Chaperones/metabolism , rab GTP-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Vesicular Transport/genetics , Gene Deletion , HeLa Cells , Humans , Lysosomal Membrane Proteins/genetics , Membrane Glycoproteins/genetics , Molecular Chaperones/genetics , Protein Interaction Domains and Motifs , rab GTP-Binding Proteins/genetics , rab7 GTP-Binding Proteins
4.
J Cell Sci ; 133(6)2020 03 16.
Article in English | MEDLINE | ID: mdl-32034082

ABSTRACT

Mutations in CLN3 are a cause of juvenile neuronal ceroid lipofuscinosis (JNCL), also known as Batten disease. Clinical manifestations include cognitive regression, progressive loss of vision and motor function, epileptic seizures and a significantly reduced lifespan. CLN3 localizes to endosomes and lysosomes, and has been implicated in intracellular trafficking and autophagy. However, the precise molecular function of CLN3 remains to be elucidated. Previous studies showed an interaction between CLN3 and Rab7A, a small GTPase that regulates several functions at late endosomes. We confirmed this interaction in live cells and found that CLN3 is required for the efficient endosome-to-TGN trafficking of the lysosomal sorting receptors because it regulates the Rab7A interaction with retromer. In cells lacking CLN3 or expressing CLN3 harbouring a disease-causing mutation, the lysosomal sorting receptors were degraded. We also demonstrated that CLN3 is required for the Rab7A-PLEKHM1 interaction, which is required for fusion of autophagosomes to lysosomes. Overall, our data provide a molecular explanation behind phenotypes observed in JNCL and give an indication of the pathogenic mechanism behind Batten disease.This article has an associated First Person interview with the first author of the paper.


Subject(s)
Membrane Glycoproteins , Molecular Chaperones , Neuronal Ceroid-Lipofuscinoses , Endosomes/genetics , Humans , Lysosomes/genetics , Membrane Glycoproteins/genetics , Molecular Chaperones/genetics , Neuronal Ceroid-Lipofuscinoses/genetics
5.
Small GTPases ; 11(3): 167-173, 2020 05.
Article in English | MEDLINE | ID: mdl-29099291

ABSTRACT

The small GTPase Rab7 is the main regulator of membrane trafficking at late endosomes. This small GTPase regulates endosome-to-trans Golgi Network trafficking of sorting receptors, membrane fusion of late endosomes to lysosomes, and autophagosomes to lysosomes during autophagy. Rab7, like all Rab GTPases, binds downstream effectors coordinating several divergent pathways. How cells regulate these interactions and downstream functions is not well understood. Recent evidence suggests that Rab7 function can be modulated by the combination of several post-translational modifications that facilitate interactions with one effector while preventing binding to another one. In this review, we discuss recent data on how phosphorylation, palmitoylation and ubiquitination modulate the ability of this small GTPase to orchestrate membrane trafficking at the late endosomes.


Subject(s)
rab GTP-Binding Proteins/metabolism , Humans , Lipoylation , Phosphorylation , Protein Processing, Post-Translational , Ubiquitination , rab7 GTP-Binding Proteins
6.
J Cell Sci ; 130(15): 2579-2590, 2017 Aug 01.
Article in English | MEDLINE | ID: mdl-28600323

ABSTRACT

Retromer is a multimeric protein complex that mediates endosome-to-trans-Golgi network (TGN) and endosome-to-plasma membrane trafficking of integral membrane proteins. Dysfunction of this complex has been linked to Alzheimer's disease and Parkinson's disease. The recruitment of retromer to endosomes is regulated by Rab7 (also known as RAB7A) to coordinate endosome-to-TGN trafficking of cargo receptor complexes. Rab7 is also required for the degradation of internalized integral membrane proteins, such as the epidermal growth factor receptor (EGFR). We found that Rab7 is palmitoylated and that this modification is not required for membrane anchoring. Palmitoylated Rab7 colocalizes efficiently with and has a higher propensity to interact with retromer than nonpalmitoylatable Rab7. Rescue of Rab7 knockout cells by expressing wild-type Rab7 restores efficient endosome-to-TGN trafficking, while rescue with nonpalmitoylatable Rab7 does not. Interestingly, Rab7 palmitoylation does not appear to be required for the degradation of EGFR or for its interaction with its effector, Rab-interacting lysosomal protein (RILP). Overall, our results indicate that Rab7 palmitoylation is required for the spatiotemporal recruitment of retromer and efficient endosome-to-TGN trafficking of the lysosomal sorting receptors.


Subject(s)
Endosomes/metabolism , Lipoylation , rab GTP-Binding Proteins/metabolism , trans-Golgi Network/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Cell Line , Endosomes/genetics , ErbB Receptors/genetics , ErbB Receptors/metabolism , Humans , Protein Transport , rab GTP-Binding Proteins/genetics , rab7 GTP-Binding Proteins , trans-Golgi Network/genetics
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