PMID- 38197533
OWN - NLM
STAT- MEDLINE
DCOM- 20240111
LR  - 20240218
IS  - 2691-1299 (Electronic)
IS  - 2691-1299 (Linking)
VI  - 4
IP  - 1
DP  - 2024 Jan
TI  - Histodenz Separation of Lysosomal Subpopulations for Analysis of 
      Chaperone-mediated Autophagy.
PG  - e950
LID - 10.1002/cpz1.950 [doi]
AB  - Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal 
      proteolysis, in which proteins are individually selected for lysosomal 
      degradation. CMA degradation targets bear a pentapeptide consensus motif that is 
      recognized by the cytosolic chaperone HSPA8 (Hsc70), which participates in the 
      trafficking of the target to the lysosomal surface. From there, it is 
      translocated into the lysosomal lumen, independent of vesicle fusion, in a 
      process dependent upon the lysosomal transmembrane protein LAMP2A. There are 
      limited tools for studying CMA in whole cells and tissues, and many of the best 
      techniques for studying CMA rely on the preparation of lysosome enriched 
      fractions. Such experiments include (1) the in vitro evaluation of CMA substrate 
      uptake activity, (2) the characterization of changes to lysosomal resident and 
      CMA regulatory proteins, and (3) lysosomal targetomics, i.e., the use of 
      quantitative proteomics to characterize lysosomal degradation targets. Previous 
      studies using discontinuous metrizamide gradients have shown that a subpopulation 
      of liver lysosomes is responsible for the majority of CMA activity ("CMA(+) "). 
      These CMA(+) lysosomes are low density and have higher levels of MTORC2 relative 
      to the "CMA(-) " lysosomes, which are high density and have higher levels of 
      MTORC1. Because of safety concerns surrounding metrizamide, however, this 
      compound is difficult to obtain, and it is impractically expensive. Here, we have 
      provided protocols for isolation of lysosomal subpopulations for CMA-related 
      analyses from mouse liver using Histodenz, a safe and affordable alternative to 
      metrizamide. Supplementary protocols show how to perform CMA activity assays with 
      appropriate statistical analysis, and how to analyze for lysosomal 
      breakage/membrane integrity. (c) 2024 The Authors. Current Protocols published by 
      Wiley Periodicals LLC. Basic Protocol: Isolation of lysosomal subpopulations from 
      mouse liver using discontinuous Histodenz gradients Alternate Protocol: Isolation 
      of lysosomes from cultured cells using discontinuous Histodenz gradients Support 
      Protocol 1: Verifying enrichment of lysosomal markers in lysosome-enriched 
      fractions Support Protocol 2: Measuring in vitro uptake of CMA substrates Support 
      Protocol 3: Measuring lysosomal membrane integrity by hexosaminidase assay.
CI  - (c) 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.
FAU - Burns, Calvin M
AU  - Burns CM
AD  - Department of Pathology, University of Michigan, Ann Arbor, Michigan.
FAU - Miller, Richard A
AU  - Miller RA
AD  - Department of Pathology, University of Michigan, Ann Arbor, Michigan.
AD  - Geriatrics Center, University of Michigan, Ann Arbor, Michigan.
FAU - Endicott, S Joseph
AU  - Endicott SJ
AUID- ORCID: 0000-0002-8237-9784
AD  - Department of Pathology, University of Michigan, Ann Arbor, Michigan.
LA  - eng
GR  - P30 AG024824/AG/NIA NIH HHS/United States
GR  - U19 AG023122/AG/NIA NIH HHS/United States
PT  - Journal Article
PL  - United States
TA  - Curr Protoc
JT  - Current protocols
JID - 101773894
RN  - RHH3W8F1CO (Metrizamide)
RN  - EC 3.2.1.52 (beta-N-Acetylhexosaminidases)
SB  - IM
MH  - Animals
MH  - Mice
MH  - *Chaperone-Mediated Autophagy
MH  - Metrizamide
MH  - Lysosomes
MH  - beta-N-Acetylhexosaminidases
MH  - Biological Assay
PMC - PMC10874119
MID - NIHMS1952206
OTO - NOTNLM
OT  - autophagy
OT  - density purification
OT  - lysosomes
OT  - organelle isolation
COIS- CONFLICT OF INTEREST STATEMENT The authors have no conflicts of interest to 
      declare.
EDAT- 2024/01/10 12:42
MHDA- 2024/01/11 07:42
PMCR- 2025/01/01
CRDT- 2024/01/10 08:13
PHST- 2025/01/01 00:00 [pmc-release]
PHST- 2024/01/11 07:42 [medline]
PHST- 2024/01/10 12:42 [pubmed]
PHST- 2024/01/10 08:13 [entrez]
AID - 10.1002/cpz1.950 [doi]
PST - ppublish
SO  - Curr Protoc. 2024 Jan;4(1):e950. doi: 10.1002/cpz1.950.