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Assays for Monitoring Autophagy in Stem Cells

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Autophagy in Stem Cell Maintenance and Differentiation

Part of the book series: Stem Cell Biology and Regenerative Medicine ((STEMCELL,volume 73))

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Abstract

Macroautophagy (Autophagy hereafter) mediates degradation of cytoplasmic components and organelles via the lysosome in all types of cells including stem cells. It is an evolutionarily conserved process which involves formation of a double membrane, the autophagosome, and is crucial for maintaining homeostasis within the cells. Numerous assays for measuring autophagy (autophagic flux) have been designed and described in detail in Klionsky et al. [72]. However, not all may be suitable for a particular stem cell type, and interpreting the assays is key to understanding autophagy in stem cells. Several researchers use drugs or transcription factors to induce stemness or in differentiation protocols, and it would be prudent to know if the treatments affect autophagy and in what way. This review is an attempt to put together the relevant assays that can be used to monitor autophagy along with the advantages and limitations of each assay. In addition, we also discuss autophagy assays that have been successfully used by researchers in a particular stem cell type.

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Abbreviations

AKT:

A serine/threonine protein kinase

AMPK:

Adenosine 5’monophosphate activated protein kinase

ASC:

Adult stem cells

Atg:

Autophagy-related gene

ATP2A/SERCA:

Sarcoplasmic/endoplasmic reticulum calcium ATPase

CCCP:

Carbonyl cyanide m-chlorophenyl hydrazone

CLEM:

Corelative light electron microscopy

CMA:

Chaperone-mediated autophagy

CQ:

Chloroquine

ESC:

Embryonic stem cells

ESCRTs:

Escort complexes

FIB-SEM:

Focused ion beam scanning electron microscopy

FIP200:

Focal adhesion kinase family interacting protein of 200 kDa

FOXO3:

Forkhead box transcription factors

FUNDC1:

Fun14 domain containing 1

FYVE:

(Cysteine-rich proteins) Fab1 YOTB Vac1 and EEA1

GABARAP:

Gamma amino butyric acid receptor-associated protein

GFP:

Green fluorescent protein

HSC:

Hematopoietic stem cells

Hsp:

Heat shock protein

LAMP:

Lysosome-associated membrane protein

LC3:

Microtubule-associated protein light chain 3

LIR:

LC3 interacting region

MAP:

Mannose-associated protein

MAPK14:

Mitogen-activated protein kinase

mCherry:

MFruits family of monomeric red fluorescent proteins

MSC:

Mesenchymal stem cells

mtHsp:

Mitochondrial Heat shock protein

mTORC:

Mammalian target of Rapamycin complex

MuSC:

Epithelial/Muscle stem cells

NBR1:

Neighbour of BRCA1 gene protein

P62:

Protein of 62 kDa

PI3K:

Phosphatidyl inositol 3 kinase

Pink1:

PTEN-induced kinase1

RB1CC1:

RB1 inducible coiled-coil protein 1

RFP:

Red fluorescent protein

SEM:

Scanning Electron Microscopy

SNARE:

Soluble NSF attachment protein receptor

SOD2:

Superoxide dismutase2

SQSTM1:

Sequestrosome

STX:

Syntaxin protein

TAX1BP1:

Tax1 binding protein1

TBK1:

TANK binding kinase1

TEM:

Transmission Electron Microscopy

TOMM:

Translocase of outer mitochondrial membrane

TSC:

Tuberous sclerosis protein

Ulk1:

Unc51-like autophagy activating kinase1

UVRAG:

UV Radiation resistance-associated gene

VDAC:

Voltage-dependent anion channel

VPS:

Vacuolar protein sorting

WIPI2:

WD repeat domain phosphoinositide interacting protein

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Acknowledgements

This work was supported by grants from DBT-Ramalingaswami Fellowship, DST-SERB grant number CRG/2020/002716 and DBT grant number BT/PR/12718/MED31/298/2015 to BS. BVS is affiliated to Savitribai Phule Pune University (SPPU), Pune, India, and is recognized by SPPU as a Ph.D. guide (Biotechnology and Zoology). The authors would also like to thank Director, ARI, and the entire ARI fraternity for facilitating our research activities in the Developmental Biology Group.

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Authors and Affiliations

  1. Developmental Biology Group, MACS-Agharkar Research Institute, Pune, India

    Aishwarya Chhatre & Bhupendra V. Shravage

  2. Department of Zoology, Savitribai Phule Pune University, Pune, India

    Bhupendra V. Shravage

  3. Department of Biotechnology, Savitribai Phule Pune University, Pune, India

    Bhupendra V. Shravage

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  1. Aishwarya Chhatre
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Correspondence to Bhupendra V. Shravage .

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  1. Developmental Biology Group, Agharkar Research Institute, Pune, Maharashtra, India

    Bhupendra V. Shravage

  2. Ottawa Hospital, Ottawa, ON, Canada

    Kursad Turksen

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Chhatre, A., Shravage, B.V. (2023). Assays for Monitoring Autophagy in Stem Cells. In: Shravage, B.V., Turksen, K. (eds) Autophagy in Stem Cell Maintenance and Differentiation. Stem Cell Biology and Regenerative Medicine, vol 73. Springer, Cham. https://doi.org/10.1007/978-3-031-17362-2_1

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