Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Summary

Lipophagy is a selective form of autophagy that involves the degradation of lipid droplets. Dysfunctions in this process are associated with cancer development. However, the precise mechanisms are not yet fully understood. This protocol describes quantitative imaging approaches to better understand the interplay between autophagy, lipid metabolism, and cancer progression.

Abstract

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This process is particularly prominent under conditions such as fasting, cellular stress, organelle damage, cellular damage, or aging of cellular components. During autophagy, a segment of the cytoplasm is enclosed within double-membrane vesicles known as autophagosomes, which then fuse with lysosomes. Following this fusion, the contents of autophagosomes undergo non-selective bulk degradation facilitated by lysosomes. However, autophagy also exhibits selective functionality, targeting specific organelles, including mitochondria, peroxisomes, lysosomes, nuclei, and lipid droplets (LDs). Lipid droplets are enclosed by a phospholipid monolayer that isolates neutral lipids from the cytoplasm, protecting cells from the harmful effects of excess sterols and free fatty acids (FFAs). Autophagy is implicated in various conditions, including neurodegenerative diseases, metabolic disorders, and cancer. Specifically, lipophagy -- the autophagy-dependent degradation of lipid droplets -- plays a crucial role in regulating intracellular FFA levels across different metabolic states. This regulation supports essential processes such as membrane synthesis, signaling molecule formation, and energy balance. Consequently, impaired lipophagy increases cellular vulnerability to death stimuli and contributes to the development of diseases such as cancer. Despite its significance, the precise mechanisms governing lipid droplet metabolism regulated by lipophagy in cancer cells remain poorly understood. This article aims to describe confocal imaging acquisition and quantitative imaging analysis protocols that enable the investigation of lipophagy associated with metabolic changes in cancer cells. The results obtained through these protocols may shed light on the intricate interplay between autophagy, lipid metabolism, and cancer progression. By elucidating these mechanisms, novel therapeutic targets may emerge for combating cancer and other metabolic-related diseases.

Introduction

Autophagy is a general term used to describe catabolic processes in which the cell transports its components to the lysosome for degradation. To date, three types of autophagy have been identified: microautophagy, macroautophagy, and chaperone-mediated autophagy^1,2,3. Macroautophagy, hereafter referred to as autophagy, is an essential pathway for regulating cellular homeostasis. Disruption of this balance can lead to the development of pathological conditions⁴.

Autophagy is a complex process that involves multiple steps. The....

Protocol

This study was conducted using epithelial adenocarcinoma HeLa cells (CCL2, ATCC). The protocol focuses on studying lipid droplets (LDs) during the induction of lipophagy in live cells to quantify the time course of LD number variation and LD-autophagosome interactions in cells expressing the wild-type (p62/SQSTM1-S182S) and two site-specific mutants of the autophagy receptor p62/SQSTM1¹⁶. Expression of a phospho-defective form (p62/SQSTM1-S182A) increases the number of LDs, while expression of a p.......

Discussion

Quantitative imaging techniques, such as confocal microscopy and image analysis protocols, have provided valuable insights into the dynamics of LDs during lipophagy^16,42,43. These technologies enable real-time visualization and quantification of LDs, allowing for the analysis of their number, size, and interactions with other organelles¹⁶. However, one of the most critical steps in this protocol is the co.......

Materials

Name	Company	Catalog Number	Comments
35 mm glass-bottom dishes	MatTek	P35G-1.5-14-C
Bafilomycin A1	Tocris	1334	200 nM
BODIPY 493/503	Invitrogen	D3922	0.5 mM
CaCl2	Merck	102378	0.1 mM
ComDet V Plugin	ImageJ	ImageJ FIJI
DAPI	Invitrogen	D1306	125 mg/mL
Dulbecco’s Modified Eagle’s Medium (DMEM)	Gibco	12800017
ES-qualified HEPES buffer	Cytiva HyClone AdvanceSTEM	SH3085101	10 mM
Etomoxir	SigmaAldrich	E1905	100 mM
Fetal Bovine Serum	Cytiva HyClone AdvanceSTEM	SH3039603	10% v/v
Forma Series II Water-Jacketed CO2 Incubator	Thermo Scientific	3111	37 °C, 5% CO2
Harmony Phenologic software	Revvity		image analysis software
HeLa cells	ATCC	CCL-2	Maintain cells at a low passage number, ideally between 8 and 10, to ensure optimal cellular characteristics.
HEPES	Merck	110110	10 mM
High-speed clinical centrifuge	DLAB	DM0412
Immersion Oil	Leica	11513859
MgCl2	Merck	814733	1 mM
Operetta CLS Live spinning-disk microscope	Revvity	HH16000020
Optical bottom 96-well plates	Thermo Scientific	165305
Paraformaldehyde	Electron Microscopy Sciences	157-8	4%v/v
penicillin/streptomycin/Amphotericin B	Biological Industries	030331b	(1000 µ/mL, 100 mg/mL, 100 mg/mL)
Phosphate-buffered saline (PBS)	Sartorius	020235A	1x
Red-phenol free DMEM	Gibco	31053028
T863	Merck	SML0539	50 mM
TCS SP8 Leica confocal microscope	Leica Microsystems
TransIT-LT1 Transfection Reagent	Mirus	MIR 2304
Triton X-100	Merck	T9284	0.20%
Trypsin/EDTA	Gibco	252000056	0.25% v/v
UNO-TEMP controller	Okolab	OK-H401-T-CONTROLLER	37 °C