Life Sciences Research for Lifelong Health

Research Summary

Research in our lab is focused on the related topics of autophagy (self eating), macroendocytosis (digestion of extracellular material) and entosis (a recently discovered form of cell cannibalism). These are 3 distinct but inter-related forms of cellular ‘eating’, which play an important role in normal biology and become deregulated during aging or disease (eg cancer).

Our work exploits a combination of molecular and cellular biology, state-of-the-art microscopy (long-term timelapse imaging, spinning disk confocal and electron microscopy) and proteomics (mass spectrometry).

Existing projects aim to define the molecular mechanisms which underlie cellular eating, with a particular focus on the emerging pathway of non-canonical autophagy. We are also investigating the intriguing relationship between entosis and cancer.

Latest Publications

Pharmacological modulators of autophagy activate a parallel noncanonical pathway driving unconventional LC3 lipidation.
Jacquin E, Leclerc-Mercier S, Judon C, Blanchard E, Fraitag S, Florey O

The modulation of canonical macroautophagy/autophagy for therapeutic benefit is an emerging strategy of medical and pharmaceutical interest. Many drugs act to inhibit autophagic flux by targeting lysosome function, while others were developed to activate the pathway. Here, we report the surprising finding that many therapeutically relevant autophagy modulators with lysosomotropic and ionophore properties, classified as inhibitors of canonical autophagy, are also capable of activating a parallel noncanonical autophagy pathway that drives MAP1LC3/LC3 lipidation on endolysosomal membranes. Further, we provide the first evidence supporting drug-induced noncanonical autophagy in vivo using the local anesthetic lidocaine and human skin biopsies. In addition, we find that several published inducers of autophagy and mitophagy are also potent activators of noncanonical autophagy. Together, our data raise important issues regarding the interpretation of LC3 lipidation data and the use of autophagy modulators, and highlight the need for a greater understanding of the functional consequences of noncanonical autophagy.

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Autophagy, , 1554-8635, 1-14, 2017

PMID: 28296541

PIKfyve Regulates Vacuole Maturation and Nutrient Recovery following Engulfment.
Krishna S, Palm W, Lee Y, Yang W, Bandyopadhyay U, Xu H, Florey O, Thompson CB, Overholtzer M

The scavenging of extracellular macromolecules by engulfment can sustain cell growth in a nutrient-depleted environment. Engulfed macromolecules are contained within vacuoles that are targeted for lysosome fusion to initiate degradation and nutrient export. We have shown that vacuoles containing engulfed material undergo mTORC1-dependent fission that redistributes degraded cargo back into the endosomal network. Here we identify the lipid kinase PIKfyve as a regulator of an alternative pathway that distributes engulfed contents in support of intracellular macromolecular synthesis during macropinocytosis, entosis, and phagocytosis. We find that PIKfyve regulates vacuole size in part through its downstream effector, the cationic transporter TRPML1. Furthermore, PIKfyve promotes recovery of nutrients from vacuoles, suggesting a potential link between PIKfyve activity and lysosomal nutrient export. During nutrient depletion, PIKfyve activity protects Ras-mutant cells from starvation-induced cell death and supports their proliferation. These data identify PIKfyve as a critical regulator of vacuole maturation and nutrient recovery during engulfment.

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Developmental cell, 38, 1878-1551, 536-47, 2016

PMID: 27623384

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy.
Russell MR, Lerner TR, Burden JJ, Nkwe DO, Pelchen-Matthews A, Domart MC, Durgan J, Weston A, Jones ML, Peddie CJ, Carzaniga R, Florey O, Marsh M, Gutierrez MG, Collinson LM

The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy and 3D image processing/analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research.

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Journal of cell science, , 1477-9137, , 2016

PMID: 27445312

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Keywords

autophagy
cell biology
entosis
macroendocytosis

entosis and the formation of a cell-in-cell structure by MCF10A cells
A) Sequence of images showing entosis and the formation of a cell-in-cell structure by MCF10A cells in suspension. Cell 1 is engulfed by Cell 2.

B) H&E staining from a human breast carcinoma, arrows point to cell-in-cell structures (taken from Biomax.us).

C) Immunofluorescent staining of b-catenin in a cell-in-cell structure from a human breast tumor.

D) Immunofluorescent staining of E-cadherin in a cell-in-cell structure from MCF10A cells.

Group Members

Latest Publications

PIKfyve Regulates Vacuole Maturation and Nutrient Recovery following Engulfment.

Krishna S, Palm W, Lee Y

Developmental cell
38 1878-1551:536-47 (2016)

PMID: 27623384

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy.

Russell MR, Lerner TR, Burden JJ

Journal of cell science
1477-9137: (2016)

PMID: 27445312

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).

Klionsky DJ, Abdelmohsen K, Abe A

Autophagy
12 1554-8635:1-222 (2016)

PMID: 26799652

V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation.

Florey O, Gammoh N, Kim SE

Autophagy
1554-8635:0 (2014)

PMID: 25484071

SOS1 and Ras regulate epithelial tight junction formation in the human airway through EMP1.

Durgan J, Tao G, Walters MS

EMBO reports
16 1469-3178:87-96 (2015)

PMID: 25394671

Competition between human cells by entosis.

Sun Q, Luo T, Ren Y

Cell research
24 1748-7838:1299-310 (2014)

PMID: 25342560

Competition between human cells by entosis.

Sun Q, Luo T, Ren Y

Cell research
24 1748-7838:1299-310 (2014)

PMID: 25342560

Interaction between FIP200 and ATG16L1 distinguishes ULK1 complex-dependent and -independent autophagy.

N Gammoh, O Florey, M Overholtzer

Nature structural & molecular biology
20 2:144-9 (2013)

DOI: 10.1038/nsmb.2475

PMID: 23262492

Autophagy proteins in macroendocytic engulfment.

O Florey, M Overholtzer

Trends in cell biology
22 7:374-80 (2012)

DOI: 10.1016/j.tcb.2012.04.005

PMID: 22608991