Life Sciences Research for Lifelong Health

Oliver Florey

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

The double life of autophagy proteins.
Florey O

Nature microbiology, 3, 2058-5276, 1334-1335, 2018

PMID: 30478385

Alpha-synuclein fibrils recruit TBK1 and OPTN to lysosomal damage sites and induce autophagy in microglial cells.
Bussi C, Peralta Ramos JM, Arroyo DS, Gallea JI, Ronchi P, Kolovou A, Wang JM, Florey O, Celej MS, Schwab Y, Ktistakis NT, Iribarren P

Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not clear and most work was done in neurons and not in microglial cells. Here we report that exogenous fibrillar but not monomeric alpha-synuclein (AS) induces autophagy in microglial cells. We extensively studied the dynamics of this response by both live-cell imaging and correlative light-electron microscopy (CLEM) and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and Optineurin (OPTN) to ubiquitinated lysosomes. In addition, we observed that LC3 recruitment to damaged lysosomes was dependent on TBK1 activity. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS.

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Journal of cell science, , 1477-9137, , 2018

PMID: 30404831

The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independently of the WD domain required for LC3-associated phagocytosis.
Rai S, Arasteh M, Jefferson M, Pearson T, Wang Y, Zhang W, Bicsak B, Divekar D, Powell PP, Nauman R, Beraza N, Carding SR, Florey O, Mayer U, Wileman T

Macroautophagy/autophagy delivers damaged proteins and organelles to lysosomes for degradation, and plays important roles in maintaining tissue homeostasis by reducing tissue damage. The translocation of LC3 to the limiting membrane of the phagophore, the precursor to the autophagosome, during autophagy provides a binding site for autophagy cargoes, and facilitates fusion with lysosomes. An autophagy-related pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosome and endosome membranes during uptake of bacterial and fungal pathogens, and targets LC3 to swollen endosomes containing particulate material or apoptotic cells. We have investigated the roles played by autophagy and LAP in vivo by exploiting the observation that the WD domain of ATG16L1 is required for LAP, but not autophagy. Mice lacking the linker and WD domains, activate autophagy, but are deficient in LAP. The LAP mice survive postnatal starvation, grow at the same rate as littermate controls, and are fertile. The liver, kidney, brain and muscle of these mice maintain levels of autophagy cargoes such as LC3 and SQSTM1/p62 similar to littermate controls, and prevent accumulation of SQSTM1 inclusions and tissue damage associated with loss of autophagy. The results suggest that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis. Further deletion of glutamate E230 in the coiled-coil domain required for WIPI2 binding produced mice with defective autophagy that survived neonatal starvation. Analysis of brain lysates suggested that interactions between WIPI2 and ATG16L1 were less critical for autophagy in the brain, which may allow a low level of autophagy to overcome neonatal lethality. Abbreviations: CCD: coiled-coil domain; CYBB/NOX2: cytochrome b-245: beta polypeptide; GPT/ALT: glutamic pyruvic transaminase: soluble; LAP: LC3-associated phagocytosis; LC3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NOD: nucleotide-binding oligomerization domain; NADPH: nicotinamide adenine dinucleotide phosphate; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing Beclin 1-interacting protein; SLE: systemic lupus erythematosus; SQSTM1/p62: sequestosome 1; TLR: toll-like receptor; TMEM: transmembrane protein; TRIM: tripartite motif-containing protein; UVRAG: UV radiation resistance associated gene; WD: tryptophan-aspartic acid; WIPI: WD 40 repeat domain: phosphoinositide interacting.

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Autophagy, , 1554-8635, 1-14, 2018

PMID: 30403914

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

Mitosis can drive cell cannibalism through entosis.

Durgan J, Tseng YY, Hamann JC

eLife
6 2050-084X: (2017)

PMID: 28693721

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