The selective autophagy adaptor p62/SQSTM1 forms phase condensates regulated by HSP27 that facilitate the clearance of damaged lysosomes via lysophagy

. Author manuscript; available in PMC: 2023 Oct 23.

Published in final edited form as: Cell Rep. 2023 Jan 25;42(2):112037. doi: 10.1016/j.celrep.2023.112037

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
ο/β-tubulin	Cell Signaling	Cat#:2148, RRID:AB_2288042
Alexa Fluor 488 goat anti-chicken IgG (H + L)	Invitrogen	Cat#: A11039, RRID: AB_2534096
Alexa Fluor 488 goat anti-rabbit IgG (H + L)	Invitrogen	Cat#: A11034, RRID: AB_2576217
Alexa Fluor 555 goat anti-chicken IgG (H + L)	Invitrogen	Cat#: A21437, RRID: AB_2535858
Alexa Fluor 594 donkey anti-sheep IgG (H + L)	Invitrogen	Cat#: A11016, RRID: AB_2535083
Alexa Fluor 594 goat anti-mouse IgG (H + L)	Invitrogen	Cat#: A11032, RRID: AB_2534091
Alexa Fluor 594 goat anti-rabbit IgG (H + L)	Invitrogen	Cat#: A11037, RRID: AB_2534095
Alexa Fluor Plus 647 goat anti-mouse IgG (H + L)	Invitrogen	Cat#: A32728, RRID: AB_2633277
Alexa Fluor 647 goat anti-rabbit IgG (H + L)	Invitrogen	Cat#: A31573, RRID: AB_2536183
Alexa Fluor 680 AffiniPure Goat anti-Mouse IgG light chain	Jackson Immuno	Cat#: 115-625-174, RRID: AB_2338937
FIP200	Proteintech	Cat#: 17250-1-AP, RRID: AB_10666428
FLAG M2	Sigma	Cat#: F3165, RRID: AB_259529
GABARAP (E1J4E)	Cell Signaling	Cat#: 13733, RRID: AB_2798306
GABARAP/L1/L2 (EPR4805)	Abcam	Cat#: ab109364, RRID: AB_10861928
Galectin-3	Santa Cruz Biotech	Cat#: sc-32790, RRID: AB_627657
Galectin-8	Abcam	Cat#: ab42879, RRID: AB_880161
GAPDH	Abcam	Cat#: ab9484, RRID: AB_307274
GFP	Abcam	Cat#: ab1218, RRID: AB_298911
GFP	Aves	Cat#: GFP-1020, RRID: AB_10000240
GFP-Booster ATTO 488	Chromotek	Cat#: gba488, RRID: AB_2631386
HaloTag	Promega	Cat#: G9281, RRID: AB_713650
HA-Tag (16B12) Alexa flour-594	Invitrogen	Cat#: A21288, RRID: AB_1500205
HSP27	Santa Cruz Biotech	Cat#: sc-13132, RRID: AB_627755
HSP27 pS15	Sigma	Cat#: SAB4300134, RRID: AB_10620369
HSP27 pS78	Cell Signaling	Cat#: 2405, RRID: AB_2120486
HSP27 pS82	Cell Signaling	Cat#: 2406, RRID: AB_2120485
HSP90 (C45G5)	Cell Signaling	Cat#: 4877T, RRID: AB_2233307
IRDye 680RD Donkey anti-Chicken	Li-Cor	Cat#: 926–68075, RRID: AB_10974977
IRDye 680RD Donkey anti-Guinea Pig	Li-Cor	Cat#: 926–68077, RRID: AB_10956079
IRDye 680RD Donkey anti-Rabbit	Li-Cor	Cat#: 926–68073, RRID: AB_10954442
IRDye 800CW Donkey anti-Mouse	Li-Cor	Cat#: 926–32212, RRID: AB_621847
IRDye 800CW Donkey anti-Rabbit	Li-Cor	Cat#: 926–32213, RRID: AB_621848
LAMP1	Abcam	Cat#: ab24170, RRID: AB_775978
LAMP1	R&D Systems	Cat#: AF4800, RRID: AB_1026176
LAMP1 Alexa Fluor 488	R&D Systems	Cat#: IC7985G, RRID:AB_2927803
LC3B	Abcam	Cat#: ab48394, RRID: AB_881433
LC3B	Novus Biologicals	Cat#: NB100-2220, RRID: AB_10003146
LAMTOR2/p14 (EPR14378)	Abcam	Cat#: ab183514, RRID: AB_2927804
mCherry	Abcam	Cat#: ab205402, RRID: AB_2722769
NDP52	Abcam	Cat#: ab68588, RRID: AB_1640255
OPTN	Abcam	Cat#: ab23666, RRID: AB_447598
Tau	Sigma	Cat#: MAB3420, RRID: AB_11212769
Tau	Abcam	Cat#: ab8763, RRID: AB_2927805
TAX1BP1 (EPR13287-B)	Abcam	Cat#: ab176572, RRID: AB_2927806
p62	American Research Products	Cat#: 03-GP62-C, RRID: AB_1542690
p62	Abcam	Cat#: ab56416, RRID: AB_945626
Bacterial and virus strains
Competent E. coli	New England Biolabs	Cat#: C3040I
Chemicals, peptides, and recombinant proteins
L-Leucyl-L-Leucine methyl ester monohydrochloride (LLOMe)	Cayman Chemicals	Cat#: 16008
Ethanol, 200 Proof	Decon Labs	Cat#: 2716
Dulbecco’s Modified Eagle’s Medium	Corning	Cat#: MT10-013-CV
Dulbecco’s Modified Eagle’s Medium	Gibco	Cat#: 11965084
Fetal Bovine Serum	HyClone	Cat#: SH3007103
GlutaMax	Thermo Fisher	Cat#: 35050061
Penicillin/streptomycin	Sigma	Cat#: P0781
Poly-L-Lysine	Sigma	Cat#: P1274
Poly-L-Ornithine	Sigma	Cat#: P3655
Neurobasal	Gibco	Cat#: 2110304
D-Glucose solution 45%	Sigma	Cat#: G8769
Sodium pyruvate	Corning	Cat#: 36017004
B27 supplement	Gibco	Cat#: 17504044
AraC	Sigma	Cat#: C6645
Minimum essential medium (MEM)	Thermo Fisher	Cat#: 11,095-072
Horse serum (heat inactivated)	Thermo Fisher	Cat#: 16,050-122
Essential 8 medium	Thermo Fisher	Cat#: A1517001
ReLeSR	Stem Cell Tech	Cat#: 05872
Accutase	Stem Cell Tech	Cat#: 07920
ROCK Inhibitor Y-27632	Selleck	Cat#: S1049
DMEM/F-12, HEPES	Thermo Fisher	Cat#: 11330032
BrainPhys Neuronal Medium	StemCell	Cat#: 05790
BDNF	PeproTech	Cat#: 450-02
NT-3	PeproTech	Cat#: 450-03
Laminin	Corning	Cat#: 354232
FuGene 6	Promega	Cat#: E269A
Lipofectamine RNAiMax	Invitrogen	Cat#: 13778150
Lipofectamine 2000	Invitrogen	Cat#: 11668030
Leibovitz’s L-15 Medium	Gibco	Cat#: 11-514-064
Viral Boost Reagent	ALSTEM	Cat#: VB100
Polybrene	Sigma	Cat#: H9268
Bouin’s Solution	Sigma	Cat#: HT10132
4% PFA in PBS	Thermo Fisher	Cat#: J19943.K2
Methanol	Thermo Fisher	Cat#: A545-1
Janelia Fluor 646-Halo ligand	Promega	Cat#: GA1120
Janelia Fluor Halo-TMR ligand	Promega	Cat#: G8251
Hoechst 33,342	Thermo Fisher	Cat#: H3570
ProLong Gold Antifade mountant	Life Technologies	Cat#: P36930
Bafilomycin A1	Sigma	Cat#: SML1661
Tween 20	Bio-Rad	Cat#: 17065
Halt Protease and Phosphatase Inhibitor Cocktail	Thermo Fisher	Cat#: 78442
10% SDS	Invitrogen	Cat#: 15,553-035
Tris Base	Thermo Fisher	Cat#: BP142-1
NaCl	Thermo Fisher	Cat#: BP358-1
KCl	Sigma	Cat#: P9541
EDTA	Invitrogen	Cat#: 15,575-038
NP-40	Sigma	Cat#: I3021
Triton X-	Sigma	Cat#: X100
Glycerol	Invitrogen	Cat#: 15,514-011
Phenylmethanesulfonyl Fluoride	Sigma	Cat#: P7626
TAME	Sigma	Cat#: 172104
Leupeptin	Sigma	Cat#: 11017128001
Pepstatin-A	Sigma	Cat#: EI10
NaPO₄	Thermo Fisher	Cat#: BP330
KH₂PO₄	Thermo Fisher	Cat#: P285
Sucrose	Thermo Fisher	Cat#: BP220
Critical commercial assays
BCA Protein Assay Kit	Thermo Fisher	Cat#: 23225
MycoAlert Detection Kit	Lonza	Cat#: LT07
Plasmid Maxi Kit	Zymo research	Cat#: D4202
Experimental models: Cell lines
HeLa-M	Andrew Peden, Cambridge Institute for Medical Research	N/A
HeLa WT	Richard Youle, National Institutes of Health	N/A
p62KO HeLa	Richard Youle, National Institutes of Health	N/A
pentaKO HeLa	Richard Youle, National Institutes of Health	N/A
HEK293	Gibco	Cat#: R70507
Experimental models: Organisms/strains
Rat: Dissociated hippocampal neuron cultures from embryonic day 18 rat brains (Sprague Dawley)	University of Pennsylvania, Neuron Culture Service Center	N/A
Mice: C57BL/6NTac WT	Taconic	Model #B6
Oligonucleotides
p62 siRNA #1	Horizon Discovery	Cat#: J-010230-05
p62 siRNA #2	Horizon Discovery	Cat#: J-010230-07
ATG5 siRNA #1	Horizon Discovery	Cat#: J-004374-09
ATG5 siRNA #2	Horizon Discovery	Cat#: L-004374-00
HSP27 siRNA #1	Horizon Discovery	Cat#: L-005269-00
HSP27 siRNA #2	Horizon Discovery	Cat#: J-005269-06
Recombinant DNA
pEGFP-C2-WT HSP27	Voss et al.⁹²	Addgene plasmid: 17,444
pEGFP-C2-3XSA (S15D/S78D/S82) HSP27	This paper	N/A
pEGFP-C2-3XSD (S15D/S78D/S82) HSP27	This paper	N/A
pEGFP-C1-Galectin-3	This paper	N/A
pFN21A-HaloTag-TAX1BP1	Promega	Catalog#: FHC02883
pEGFP-C3-TAX1BP1	Moore and Holzbaur,⁹³	N/A
pEGFP-C1-NBR1	Peter Kim Lab, The Hospital for Sick Children, University of Toronto	N/A
pEGFP-C1	ClonTech	Cat#: V012024
pHaloTag-N1	Promega	Cat#: G7721
pHaloTag-DFCPI	Stavoe et al.⁹⁴	N/A
pHaloTag-WIPI2B	Stavoe et al.⁹⁴	N/A
pLAMP1-KillerRed (EGFP bb)	This paper	N/A
pLAMP1-HTC	This paper	N/A
pTagBFP-N-LAMP2	This paper	N/A
pLJC5-Tmem192-3xHA	Abu-Remaileh et al.⁴¹	Addgene plasmid: 102,930
pRK5-FLAG-LAMTOR2	Bar-Peled et al.⁹⁵	Addgene plasmid: 42,330
pmCherry-LAMTOR2	This paper	N/A
pmCherry-LC3B	Fu et al. 2014⁹⁶	N/A
pHaloTag-LC3B	This paper	N/A
pmCherry-C1	Sascha Martens, University of Vienna	N/A
pmCherry-C1-WT-p62	Sascha Martens, University of Vienna	N/A
pmCherry-C1-p62ΔUBA	Sascha Martens, University of Vienna	N/A
pmCherry-C1-p62ΔPB1	Sascha Martens, University of Vienna	N/A
pmCherry-C1-p62 LIR^AAAA	Sascha Martens, University of Vienna	N/A
pmCherry-C1-p62 K7A/D69A	Sascha Martens, University of Vienna	N/A
pmCherry-C1-p62 G425R	This paper	N/A
pmCherry-C1-p62 L341V	This paper	N/A
pmCherry-C1-p62 P392L	This paper	N/A
pEGFP-C1-hWT-p62	This paper	N/A
pMXs-puro-eGFP-p62	Itakura et al.⁹⁷	Addgene plasmid: 38,277
pHaloTag-p62	This paper	N/A
pPGK-LAMP1mNeon	Michael Ward, National Institutes of Health	N/A
pCMV-VSV-G	Bob Weinberg, Whitehead institute	Addgene plasmid: 8454
psPAX2	Didier Trono, EPFL	Addgene plasmid: 12,260
Software and algorithms
Prism version 9.2.0	Graphpad	N/A
Fiji Image Analysis	ImageJ	https://imagej.net/software/fiji/
Volocity	PerkinElmer	https://www.perkinelmer.com/
Visiview	Visitron Systems	https://www.visitron.de/
Ilastik	Berg et al.⁹⁸	https://www.ilastik.org/
Other
Glass-bottom tissue culture dishes	MatTek	Cat#: P35G-1.5-20-C
Glass coverslips	Electron Microscopy Science, Fisher	Cat#: 50-948-975
DynaBeads Protein G	Thermo Fisher	Cat#: 10003D
Pierce Anti-HA Magnetic beads	Thermo Fisher	Cat#: 88837
Immobilon-FL PVDF membranes	Sigma	Cat#: IPFL00010
TrueBlack WB Blocking Buffer	Biotum	Cat#: 23013
EveryBlot Blocking Buffer	Bio-Rad	Cat#: 12010020