REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
rabbit anti-β-galactosidase	Cappel	Cat#0855976
rabbit anti-pH3	Millipore	Cat#06–570; RRID:AB_310177
mouse anti-GFP	Invitrogen	Cat#A11120; RRID:AB_221568
rabbit anti-GFP	Invitrogen	Cat#A6455; RRID:AB_221570
mouse anti-Flag	Sigma	Cat#F3165; RRID:AB_259529
rabbit anti-cleaved-caspase 3	Cell Signaling	Cat#9661; RRID:AB_2341188
rabbit anti-Tsp2A	Izumi et al., 2016	N/A
rabbit anti-Mesh	Izumi et al., 2016	N/A
rabbit anti-Ssk	Izumi et al., 2016	N/A
rabbit anti-Cora	DSHB	Cat#C615.16; RRID:AB_1161644
mouse anti-Arm	DSHB	Cat#N27A1; RRID:AB_528089
rabbit anti-Lgl	Santa Cruz	Cat#sc-98260; RRID:AB_1564606
rabbit anti-Pdm1	Xiaohang Yang	N/A
mouse anti-Pros	DSHB	Cat#MR1A; RRID:AB_528440
mouse anti-Hrs	DSHB	Cat#27–4; RRID:AB_2618261
mouse anti-Rab7	DSHB	Cat#Rab7; RRID:AB_2722471
guinea pig anti-ex	Richard Fehon	Maitra et al., 2006
rabbit anti-aPKC	Santa Cruz	Cat#sc-216
rabbit anti-Rack1	Julie Kadrmas	Kadrmas et al., 2007
rat anti-HA (clone 3F10)	Sigma	Cat#11867423001; RRID:AB_390919
donkey anti–rabbit IgG, Alexa Fluor 488	Invitrogen	Cat#A21206; RRID:AB_141708
donkey anti–rabbit IgG, Alexa Fluor 594	Invitrogen	Cat#A21207; RRID:AB_141637
donkey anti–mouse IgG, Alexa Fluor 488	Invitrogen	Cat#A21202; RRID:AB_141607
donkey anti–mouse IgG, Alexa Fluor 594	Invitrogen	Cat#A21203; RRID:AB_141633
donkey anti–guinea pig IgG, Alexa Fluor 594	Jackson ImmunoResearch Labs	RRID:AB_2340474
goat anti–rat IgG, Alexa Fluor 488	Invitrogen	Cat#A11006; RRID:AB_141373
rabbit anti-GFP	Abcam	Cat#6556; RRID:AB_305564
rabbit anti-GFP	Invitrogen	Cat#A6455
GFP-Trap agarose beads	Bulldog Biotechnology	Cat#GTA100
Chemicals, Peptides, and Recombinant Proteins
Paraformaldehyde 16% solution	Electron Microscopy Sciences	Cat#15710
PBS pH 7.4	GIBCO	Cat#10010023
Bleomycin	Calbiochem	Cat#203408
blue dye no.1	Spectrum Chemical	Cat#3844–45-9
Texas Red-avidin	Invitrogen	Cat#A820
Lysotracker Red DND-99	Invitrogen	Cat#L-7528
TRIzol Reagent	Invitrogen	Cat#15596018
RNase-Free DNase I Set	QIAGEN	Cat#79254
iScript Reverse Transcription Supermix	Bio-Rad	Cat#1708896
iQ SYBR Green Supermix	Bio-Rad	Cat#1708880
Cyclohexamide	Sigma	Cat#C7698
RNaseOUT ribonuclease inhibitor	Invitrogen	Cat#10777019
cOmplete Protease Inhibitor, EDTA-free	Roche	Cat#11836170001
Gateway LR Clonase II Enzyme mix	Invitrogen	Cat#11791–020
pENTR/D-TOPO Cloning Kit	Invitrogen	Cat#K2400–20
Effectene Transfection Reagent	QIAGEN	Cat#301425
GIBCO Schneider’s Drosophila Sterile Medium	Thermo Fisher	Cat#21720024
Fetal Bovine Serum	Thermo Fisher	Cat#10437028
Protease and phosphatase inhibitor cocktail	Pierce	Cat#78440
Critical Commercial Assays
RNeasy Mini kit	QIAGEN	Cat#74104
RNeasy MinElute Cleanup kit	QIAGEN	Cat#74204
4%–20% Mini-PROTEAN TGX Precast Protein Gels	Bio-Rad	Cat#4561096
Experimentl Models: Cell Lines
D. melanogaster: Cell line S2R+	Perrimon lab	N/A
Experimental Models: Organisms/Strains
D. melanogaster: UAS-Tsp2A sgRNA	This paper	N/A
D. melanogaster: UAS-FH-Tsp2A	This paper	N/A
D. melanogaster: UAS-GFP-Tsp2A	This paper	N/A
D. melanogaster: Dl-lacZ	BDSC	11651
D. melanogaster: y v; attP2 (Ctrlbl)	BDSC	36303
D. melanogaster: UAS-Luc RNAi	BDSC	31603
D. melanogaster: UAS-Tsp2A RNAi	BDSC	40899
D. melanogaster: UAS-Luc	BDSC	35789
D. melanogaster: FRT19A	BDSC	1744
D. melanogaster: UAS-bsk RNAi	BDSC	31323, 32977 (#2)
D. melanogaster: UAS-bskDN	BDSC	6409
D. melanogaster: UAS-GFP-RpL10Ab	BDSC	42683
D. melanogaster: UAS-rpr	BDSC	5823
D. melanogaster: UAS-Hepca	BDSC	6406
D. melanogaster: UAS-CD8-GFP	BDSC	32186
D. melanogaster: UAS-Rab5ca	BDSC	43335
D. melanogaster: UAS-YFP-Rab5ca	BDSC	9773
D. melanogaster: UAS-Rab7ca	BDSC	42707
D. melanogaster: UAS-YFP-Rab11ca	BDSC	50783
D. melanogaster: UAS-Rab5DN	BDSC	42704
D. melanogaster: UAS-YFP-Rab7DN	BDSC	9778
D. melanogaster: UAS-YFP-Rab11DN	BDSC	23261
D. melanogaster: UAS-Rab5 RNAi	BDSC	30518
D. melanogaster: UAS-Rab7 RNAi	BDSC	27051
D. melanogaster: UAS-Rab11 RNAi	BDSC	42709, 27730 (#2)
D. melanogaster: UAS-arm RNAi	BDSC	31305, 31304 (#2)
D. melanogaster: UAS-Lgl RNAi	BDSC	35773, 38989 (#2)
D. melanogaster: UAS-cora RNAi	BDSC	28933, 51845 (#2)
D. melanogaster: UAS-Gli RNAi	BDSC	38284, 58115 (#2)
D. melanogaster: UAS-aPKC RNAi	BDSC	34332, 35001 (#2)
D. melanogaster: UAS-Rack1 RNAi	BDSC	34694
D. melanogaster: UAS-aPKCDN	BDSC	51673
D. melanogaster: UAS-Yki3SA	BDSC	28817
D. melanogaster: UAS-Yki RNAi	BDSC	31965
D. melanogaster: UAS-hpo	BDSC	44254
D. melanogaster: UAS-dome RNAi	BDSC	32860
D. melanogaster: UAS-Stat92E RNAi	BDSC	33637
D. melanogaster: hop2	BDSC	6032
D. melanogaster: hop27	BDSC	8493
D. melanogaster: y w; attP (Ctrlv)	VDRC	60100
D. melanogaster: UAS-Mesh RNAiv	VDRC	108297
D. melanogaster: UAS-Ssk RNAiv	VDRC	11906, 105193 (#2)
D. melanogaster: UAS-Yki RNAiv	VDRC	104523
D. melanogaster: UAS-Tsp2A RNAiN	NIG	11415R-2
D. melanogaster: UAS-Rack1–3xHA	FlyORF	F001043
D. melanogaster: UAS-aPKC-3xHA	FlyORF	F000876
D. melanogaster: y w (Ctrlyw)	Perrimon lab	N/A
D. melanogaster: w1118 (Ctrlw)	Perrimon lab	N/A
D. melanogaster: Diap1-lacZ/TM6B	Karpowicz et al., 2010	N/A
D. melanogaster: UAS-p35 (on 3rd chromosome)	Perrimon lab	N/A
D. melanogaster: esgGal4 UAS-GFP tubGal80ts (EGT)	Micchelli and Perrimon, 2006	N/A
D. melanogaster: EGT; UAS-Cas9 (attP2)	Perrimon lab	N/A
D. melanogaster: esgGal4 tubGal80ts (esgts)	Perrimon lab	N/A
D. melanogaster: Myo1AGal4 tubGal80ts (Myo1Ats)	Perrimon lab	N/A
D. melanogaster: tubGal80ts; 24BGal4 (24Bts)	Perrimon lab	N/A
D. melanogaster: tubGal80ts; DaGal4 (Dats)	Perrimon lab	N/A
D. melanogaster: tubGal80ts; DlGal4 (Dlts)	Zeng et al., 2010	N/A
D. melanogaster: tubGal80ts; Su(H)Gbe-Gal4 (Su(H)ts)	Zeng et al., 2010	N/A
D. melanogaster: Su(H)Gbe-Gal4, UAS-CD8-GFP; tubGal80ts (SGT)	Zeng et al., 2010	N/A
D. melanogaster: tubGal80ts; ProsGal4 (Prosts)	Perrimon lab	N/A
D. melanogaster: hsFlp tubGal80 FRT19A/FM7; tubGal4 UAS-mCD8::GFP/TM3	Choi et al., 2011	N/A
Oligonucleotides
Tsp2A sgRNAs	This paper	See Table S3
RT-qPCR primers	This paper	See Table S3
Recombinant DNA
cDNA RE51204	Drosophila Genomics Resource Center	9398
cDNA FI03288	Drosophila Genomics Resource Center	1621950
cDNA RE74715	Drosophila Genomics Resource Center	10113
pENTR/D-TOPO-Tsp2A	This paper	N/A
pENTR/D-TOPO-aPKC	This paper	N/A
pENTR/D-TOPO-Rack1	This paper	N/A
pAGW-Tsp2A	This paper	N/A
pAFW-Tsp2A	This paper	N/A
pAGW-aPKC	This paper	N/A
pAFW-aPKC	This paper	N/A
pAGW-Rack1	This paper	N/A
pAFW-Rack1	This paper	N/A
Software and Algorithms
ZEN 2 lite	Zeiss	N/A
ImageJ/Fiji	Schindelin et al., 2012	N/A
CFX Manager	Bio-Rad	N/A
STREAM	Chen et al., 2018	N/A
Prism	GraphPad Software	N/A
Other
Zeiss LSM780 microscope	Zeiss	N/A
CFX96 Real-Time System	Bio-Rad	N/A
BZ-9000 Fluorescence Microscope	Keyence	N/A
1200-EX transmission electron microscope	JEOL	N/A