Parkhurst Lab

Parkhurst Lab

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Reagents

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Parkhurst Lab reagents available from the Development Studies Hybridoma Bank

Antigen Monoclonal Line Species Reference  
Pavarotti (Pav) O8 fly 20 RRID: AB_2936338
Pavarotti (Pav) L24 fly 20 RRID: AB_2936339   
Rho1 P1D9 fly 3 RRID: AB_528263 
Wash P3H3 fly 8, 12 RRID: AB_2618389
Wash P4C9 fly 12 RRID: AB_2618391 
WASP P5E1 fly 12 RRID: AB_2618392 
WASP P3B1 fly 12 RRID: AB_2618388 
SCAR P1C1 fly 12 RRID: AB_2618386 
SCAR P1C8 fly 12  
Whamy P4A8 fly 12 RRID: AB_2618390 
Whamy P1D1 fly 12  
p120catenin P1B2 fly 3, unpublished RRID: AB_10660833 
p120catenin P4B2 fly 3, unpublished RRID: AB_2088073 
dSir2 P2E2 fly 4, unpublished RRID: AB_1553776 
dSir2 P4A10 fly 4, unpublished RRID: AB_1553778 

Parkhurst Lab reagents available from the Bloomington Drosophila Stock Center

 

Stock # Genotype Allele type | insertion chromosome Reference

wimp (RpII140 – RNA Polymerase II subunit)
5874 wimp, rucuca/TM3, Sb antimorphic allele 1

Rho1 GTPase
9477 w*; b pr cn Rho1[1B] px sp/CyO
 null allele 2,3,5

Cdc42  [R186C point mutation]
[Cdc42 point mutation (Cdc42(R186C)) under control of the UASp promoter (inducible)]
[This is a dominant negative Cdc42 mutation that gives similar phenotypes in flies to that observed in the equivalent human mutations.]
90925 w*  P{w+, UASp-Cdc42(R186C)}11 X 15
90926 w*; P{w+, UASp-Cdc42(R186C)}26/CyO 2nd 15
90927 w*; P{w+, UASp-Cdc42(R186C)}37 3rd 15

washout  (Wiskott-Aldrich Syndrome family protein)
79220 w*; wash[Delta185] null allele,background lethal removed 14
28285 w*; wash[Delta185]/CyO
 null allele 6,9


Sqh-mScarlet
[This is a CRISPR knock-in of mScarlet-i into the Spaghetti squash locus (C-terminal fusion)]
94929 TI{TI}sqhmScarlet-I ; MKRS/TM6B CRISPR knock-in (X chr) 19

sqh gRNA
[gRNA for sqh knock-in into pCFD5 vector]
94930 w*;  P{U6:3-sqh.gRNA.pCFD5}attp2 gRNA insertion on 3rd 19

degringolade
35525 w*; dgrn[DK]/TM3, Sb null allele 9

"Red" balancers (sChFP : sqh driven, ChFP protein)
35522 FM7a, P{w+; sChFP} X 11
35523 w*; Sco/CyO, P{w+, sChFP} 2nd 11
35524 w*; Gl/TM3, Sb P{w+, sChFP}     3rd 11


photoconvertible actin binding domain
 
[sM3MCA : sqh driven, Maple3 fluorescent protein, moesin α-helical-coiled and actin binding site]  
94924 w*;  P{w+, sM3MCA}12  X 19
94925 w*;  P{w+, sM3MCA}20/CyO; MKRS/TM3,Ser 2nd 19
94926 w*;  Kr/CyO; P{w+, sSM3MCA}33  3rd 19

actin binding domain

[sStMCA : sqh driven, Scarlet fluorescent protein, moesin α-helical-coiled and actin binding site]
[This Scarlet version gives much brighter fluorescence and less background than mCherry or Kate2. This reporter also recognizes nuclear actin.]
90928 w*  P{w+, sStMCA}10 X 16
90929 w*;  P{w+, sStMCA}20 2nd 16
90930 w*;  P{w+, sStMCA}20 3rd 16

actin binding domain

(sChMCA : sqh driven, ChFP protein, moesin α-helical-coiled and actin binding site)
35519  w* P{w+, sChMCA}#12 X 10
35520 w*; P{w+, sChMCA}#22 2nd 10
35521 w*; P{w+, sChMCA}#31 3rd 10

actin binding domain

(sK2MCA: sqh driven, Kate2 fluorescent protein, moesin α-helical-coiled and actin binding site)
(Kate2 is supposed to be far red.  It isn’t in flies, but it is better separated from GFP than CherryFP (giving less background fluorescence)).
76268 w* P{w+, SK2MCA}#12 X 14
76269 w*;  P{w+, sK2MCA}#12 2nd 14
76270 w*;  P{w+, sK2MCA}#12 3rd 14

actin (inducible RFP fusion protein)
24777 P{UASp-RFP.actin}13, w* X 7
24778 w*; P{UASp-RFP.actin}29 2nd 7
24779 w*; P{UASp-RFP.actin}38 3rd 7

GCaMP6s

[This is a constitutive version of the GCaMP6s calcium reporter that works very well in embryos.]
91362 w[*]; P{w[+mC]=sqh-GCaMP6s]101.1 sqh-GCaMP6s]101.2 X    (two copies of transgene) 17
91363 w[*]; P{w[+mC]=sqh-GCaMP6s}20 2nd  (one copy of transgene) 17
91364 w[*]; P{w[+mC]=sqh-GCaMP6s]305.1 sqh-GCaMP6s]305.2 3rd  (two copies of transgene) 17

GCaMP6s

[This is an inducible (UAS) version of the GCaMP6s calcium reporter in the pUASp vector (works well in the germline and embryo).]
91365 w[*]; P{w[+mC]=UASp-GCaMP6s}20/CyO 2nd  
91366 w[*]; P{w[+mC]=UASp-GCaMP6s}30/TM3, Sb 3rd  

Wash

[GFP fusion protein under control of the UASp promoter (inducible)]
81639 w*; P{w[+mC]=UASp-GFP-Wash}15 X  
81640 w*; P{w[+mC]=UASp-GFP-Wash}26 2nd  
81641 w*; P{w[+mC]=UASp-GFP-Wash}35 3rd  

Wash

[CherryFP fusion protein under control of the UASp promoter (inducible)]
81642 w*; P{w[+mC]=UASp-ChFP-Wash}10 2nd 18
81643 w*; P{w[+mC]=UASp-ChFP-Wash}31 3rd 18

Wash

[super folder GFP fusion protein under the control of the endogenous Wash promoter]
81644 w*; P{w[+mC]=Wash-sfGFP-Wash}104 2nd 14

SCAR
[GFP-SCAR fusion protein under control of endogenous SCAR promoter]
94927 w*;  P{GFP- SCAR }20; Dr/TM3,Sb 2nd 19
94928 w*;  Sp/CyO; P{GFP- SCAR }30 3rd 19

Rho1 GTPase 

(GFP fusion protein under control of endogenous Rho1 promoter)
9527 P{Rho1.GFP}, w* X 5
9528 w*; P{Rho1.GFP}        2nd 5
24762 w*; P{Rho1.GFP}30 3rd 5

Rho1 GTPase 

(ChFP fusion protein under control of endogenous Rho1 promoter)
52280 P{w[+mC]=mChFP-Rho1}10, w[*] X 13
52281 w[*]; P{w[+mC]=mChFP-Rho1}21     
 2nd 13
52282 w[*]; sna[Sco]/CyO; P{w[+mC]=mChFP-Rho1}31 3rd 13

Cdc42 GTPase 

(ChFP fusion protein under the control of the Sqh promoter)
42236 w*;  P{w[+mC]=sqh-ChFP-Cdc42}23 2nd 11
42237 w*;  P{w[+mC]=sqh-ChFP-Cdc42}3 3rd 11

Rac1 GTPase 

(GFP fusion protein under control of endogenous Rac1 promoter)
52283  
 P{w[+mC]=GFP-Rac1}10, w[*] X 13
52284 w[*]; P{w[+mC]=GFP-Rac1}20 2nd
 13
52285 w[*]; P{w[+mC]=GFP-Rac1}30 3rd
 13

Rac1 GTPase 

(CherryFP fusion protein under control of endogenous Rac1 promoter)
76266
 w[*];  P{w[+mC]=ChFP-Rac1}21 2nd 14
76267 w[*];  P{w[+mC]=ChFP-Rac1}30 3rd
 14

Rac2 GTPase 

(GFP fusion protein under control of endogenous Rac2 promoter)
52286
 w[*]; P{w[+mC]=GFP-Rac2}21 2nd 13
52287 w[*]; P{w[+mC]=GFP-Rac2}31 3rd
 13

Tumbleweed

(super folder GFP fusion protein under the control of the Sqh promoter)
76264
 w*;  P{w[+mC]=sqh-sfGFP-Tum}20 2nd 14
76265 w*;  P{w[+mC]=sqh-sfGFP-Tum}30 3rd
 14

Pebble

(GFP fusion protein under the control of the Sqh promoter)
76257
 w* P{w[+mC]=sqh-Pbl-eGFP}10 X 14
76258 w*;  P{w[+mC]=sqh-Pbl-eGFP}30 3rd
 14

RhoGEF2 

(super folder GFP fusion protein under the control of the Sqh promoter)
76259
 w* P{w[+mC]=sqh-sfGFP-RhoGEF2}10 X 14
76260 w*;  P{w[+mC]=sqh-sfGFP-RhoGEF2}30 3rd
 14

RhoGEF3  

(CherryFP fusion protein under control of the UASp promoter (inducible))
76261
 w*; P{w[+mC]=UASp-ChFP-RhoGEF3}30 3rd 14

RhoGEF3

(super folder GFP fusion protein under control of the UASp promoter (inducible))
76262
 w*; P{w[+mC]=UASp-sfGFP-RhoGEF3}20 2nd 14
76263 w*; P{w[+mC]=UASp-sfGFP-RhoGEF3}30 3rd
 14

RhoGEF2 RNAi

(21nt shRNA from RhoGEF2 cloned into WALLIUM22; 99% knockdown by qPCR)
76255 M{w[+mC]=UAS-RhoGEF2.shRNA}ZH-86Fb 3rd
 14

RhoGEF3 RNAi

(21nt shRNA from RhoGEF3 cloned into WALLIUM22; 92% knockdown by qPCR)
76256 M{w[+mC]=UAS-RhoGEF3.shRNA}ZH-86Fb 3rd
 14

Rok

(GFP fusion protein under control of Sqh promoter)
52288
 P{w[+mC]=sqh-GFP-rok}10, w[*] X 13
52289 w[*]; P{w[+mC]=sqh-GFP-rok}30 3rd
 13

Pak1

(GFP fusion protein under control of the UASp promoter (inducible)
52299
 w[*]; P{w[+mC]=UASp-GFP-Pak1}300 3rd 13

Sticky

[super folder GFP fusion protein under the control of the Sqh promoter (constitutive)]
81645 w*; P{w[+mC]=sqh-sfGFP-Sti}10 X  
81646 w*; P{w[+mC]=sqh-sfGFP-Sti}20 2nd  
81647 w*; P{w[+mC]=sqh-sfGFP-Sti}32 3rd  

Sticky

[CherryFP fusion protein under control of the UASp promoter (inducible)]
81648 w*; P{w[+mC]=UASp-ChFP-Sti}20/CyO 2nd  
81649 w*; P{w[+mC]=UASp-ChFP-Sti}30/TM3 3rd  

Rho family GTPase activity biosensors

(reporters for active Rho1, Rac, and/or Cdc42).

RokRBD

GFP fusion to Rok’s Rho1/Rac binding domain (RBD) expressed under control of the UASp promoter (inducible).
52290
 w[*]; P{w[+mC]=UASp-rok.RBD-GFP}30 3rd 13

DiaRBD

GFP fusion to Diaphanous’s Rho1 binding domain (RBD) expressed under control of the UASp promoter (inducible).
52291
 w[*]; P{w[+mC]=UASp-dia.RBD-GFP}20 2nd 13
52292
 w[*]; P{w[+mC]=UASp-dia.RBD-GFP}37 3rd 13

CapuRBD

GFP fusion to Cappuccino’s Rho1 binding domain (RBD) expressed under control of the UASp promoter (inducible).
52293
 w[*]; P{w[+mC]=UASp-capu.RBD-GFP}26 2nd 13
52294
 w[*]; P{w[+mC]=UASp-capu.RBD-GFP}34 3rd 13

WashRBD

GFP fusion to Wash’s Rho1 binding domain (RBD) expressed under control of the UASp promoter (inducible).
52295 w[*]; P{w[+mC]=UASp-wash.RBD-GFP}25 2nd 13
52296 w[*]; P{w[+mC]=UASp-wash.RBD-GFP}34 3rd 13

PknRBD

GFP fusion to Pkn’s Rho/Rac binding domain (RBD) expressed under control of the Sqh promoter (constitutive).
52297 w[*]; P{w[+mC]=sqh-Pkn.RBD.G58A-eGFP}212a
and P{sqh-Pkn.RBD.G58A-eGFP}212b		 2nd 13
52298 w[*]; P{w[+mC]=sqh-Pkn.RBD.G58A-eGFP}312a
and P{sqh-Pkn.RBD.G58A-eGFP}312b		 3rd 13

WASpRBD

GFP fusion to WASp’s Cdc42 binding domain (RBD) expressed under control of the Sqh promoter (constitutive).
56745 w[*]; P{w[+mC]=sqh-WASp.RBD-GFP}278a
P{sqh-WASp.RBD-GFP}278b/CyO		 2nd 13
56746 w[*]; P{w[+mC]=sqh-WASp.RBD-GFP}378a
P{sqh-WASp.RBD-GFP}378b		 3rd 13

Pak1RBD

GFP fusion to Pak1’s Rac/Cdc42 binding domain (RBD) expressed under control of the Sqh promoter (constitutive) or under the control of the UASp promoter (conditional).
56549 w[*]; P{w[+mC]=sqh-Pak1.RBD-GFP}21 2nd 13
56550 w[*]; P{w[+mC]=sqh-Pak1.RBD-GFP}31/TM3, Sb[1] 3rd 13
56548 w[*]; P{w[+mC]=UASp-Pak.RBD-GFP}30/TM3, Sb[1] 3rd 13

Pak3RBD

GFP fusion to Pak3’s Rac/Cdc42 binding domain (RBD) expressed under control of the Sqh promoter (constitutive).
52303 w[*]; P{w[+mC]=sqh-Pak3.RBD-GFP}20 2nd 13
52304 w[*]; P{w[+mC]=sqh-Pak3.RBD-GFP}30 3rd 13

Cappuccino

(inducible GFP fusion protein)
24763 P{UASp-GFP.Capu}10, w* X 5
24764 w*; P{UASp-GFP.Capu}20 2nd 5

Spire – C isoform

(inducible GFP fusion protein)
24765 w*; P{UASp-GFP.SpireC}21 2nd 5
24766 w*; P{UASp-GFP.SpireC}32 3rd 5

Spire – D isoform

(inducible GFP fusion protein)
24767 w*; P{UASp-GFP.SpireD}30 3rd 5

sisyphus, unconventional myosin 10A (myosin XV homolog)

(inducible RFP or GFP fusion proteins)
24780 P{UASp-Myo10A.GFP}10, w* X 7
24781 w*; P{UASp-Myo10A.GFP}20 2nd 7
24782 w*; P{UASp-Myo10A.GFP}30         3rd 7
24783 P{UASp-Myo10A.mRFP}11, w*  X 7
24784 w*; P{UASp-Myo10A.mRFP}22      2nd 7
24785 w*; P{UASp-Myo10A.mRFP}33  3rd 7

References Cited

  1. Parkhurst SM and Ish-Horowicz D (1991). wimp, a dominant maternal-effect mutation, reduces transcription of a specific subset of segmentation genes in Drosophila.  Genes Dev. 5, 341-357.
  2. Magie CR, Meyer M, Gorsuch M and Parkhurst SM (1999). Mutations in the Rho1 small GTPase disrupt morphogenesis and segmentation during early Drosophila development.  Development 126, 5353-5364.
  3. Magie CR, Pinto-Santini D and Parkhurst SM (2002). Rho1 interacts with p120ctn and alpha-catenin, and regulates cadherin-based adherens junction formation during Drosophila development.  Development 129, 3771-3782.
  4. Rosenberg MI and Parkhurst SM (2002). Drosophila Sir2 is required for heterochromatic silencing and function of euchromatic Hairy/E(spl) family bHLH repressors in segmentation and sex determination.  Cell 109, 447-458.
  5. Rosales-Nieves AE, Johndrow JE, Keller LC, Magie CR, Pinto-Santini D and Parkhurst SM (2006). Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire, and Cappuccino.  Nature Cell Biol. 8, 367-376.
  6. Linardopoulou EV, Parghi SS, Friedman C, Osborn GE, Parkhurst SM and Trask BJ (2007). Human subtelomeric WASH genes encode a new subclass of the WASP family. PLoS Genetics 3, e237.
  7. Liu R, Woolner S, Johndrow JE, Metzger D, Flores A and Parkhurst SM (2008). Sisyphus, the Drosophila myosin XV homolog, traffics within filopodia transporting key sensory and adhesion cargos. Development 135, 53-63.
  8. Liu R, Abreu-Blanco MT, Barry KC, Linardopoulou EV, Osborn GE and Parkhurst SM (2009). Wash functions downstream of Rho and links linear and branched action nucleation factors.  Development 136, 2849-2860.
  9. Barry KC, Abed M, Kenyagin D, Werwie TR, Boico O, Orian A and Parkhurst SM (2011). The Drosophila STUbL protein Degringolade limits HES function during embryogenesis. Development 138, 1759-1769.
  10. Abreu-Blanco MT, Verboon JM and Parkhurst SM (2011). Cell wound repair in Drosophila occurs through three distinct phases of membrane and cytoskeletal remodeling. J. Cell Biol. 193, 455-464.
  11. Abreu-Blanco MT, Verboon JM, Liu R, Watts JJ and Parkhurst SM (2012). Drosophila embryos close epithelial wounds using a combination of cellular protrusions and an actomyosin purse string. J. Cell Sci., 125, 5984-5997. 
  12. Rodriguez-Mesa E, Abreu-Blanco MT, Rosales-Nieves AE and Parkhurst SM (2012). Developmental expression of Drosophila Wiskott Aldrich Syndrome family proteins. Dev. Dyn. 241, 608-626
  13. Abreu-Blanco MT, Verboon JM and Parkhurst SM (2014). Coordination of Rho family GTPase activities to orchestrate cytoskeleton responses during cell wound repair. Curr. Biol., 24: 144-155.
  14. Verboon JM, Decker JR, Nakamura M and Parkhurst SM. Wash exhibits context dependent phenotypes and, along with the WASH Regulatory Complex, regulates Drosophila oogenesis. J. Cell Sci. 131(8), pii: jcs211573. doi: 10.1242/jcs.211573 (2018).   Erratum in: J. Cell Sci. 131(9), pii: jcs219212. doi: 10.1242/jcs.219212 (2018)
  15. Verboon JM, Mahmut D, Kim AR, Nakamura M, Abdulhay NJ, Nandakumar SK, Gupta N, Akie TE, Geddis AE, Manes B, Kapp ME, Hofmann I, Gabriel SB, Klein DE, Williams DA, Frangoul HA, Parkhurst SM, Crane GM, Cantor AB and Sankaran VG. (2020). Infantile Myelofibrosis and Myeloproliferation with CDC42 Dysfunction. J. Clin. Immunol. 40(4):554-566.  https://doi.org/10.1007/s10875-020-00778-7.
  16. Nakamura M, Verboon JM, Prentiss CL and Parkhurst SM (2020). The kinesin-like protein Pavarotti functions non-canonically to regulate actin dynamics. J. Cell Biol. 219(9):e201912117.  doi: 10.1083/jcb.201912117. PMID: 32673395.
  17. Nakamura M, Verboon JM, Allen TE, Abreu-Blanco MT, Liu R, Dominguez AN, Delrow JJ and Parkhurst SM (2020). Autocrine insulin pathway signaling regulates actin dynamics in cell wound repair. PLoS Genetics 16(12): e1009186. https://doi.org/10.1371/journal.pgen.1009186 
  18. Verboon JM, Rahe TK, Rodriguez-Mesa E and Parkhurst SM (2015). Wash functions downstream of Rho1 GTPase in a subset of Drosophila immune cell developmental migrations. Mol. Biol. Cell 26(9),1665-1674. doi:10.1091/mbc.E14-08-1266
  19. Hui J, Nakamura M, Dubrulle J and Parkhurst SM (2022). Coordinated efforts of different actin filament populations are needed for optimal cell wound repair. Mol. Biol. Cell 34(3):ar15. doi: 10.1091/mbc.E22-05-0155.
  20. Davidson KA, Nakamura M, Verboon JM and Parkhurst SM (2023). The Centralspindlin proteins Pavarotti and Tumbleweed function in Nuclear Envelope budding. J. Cell Biol. 222 (8): e202211074.  https://doi.org/10.1083/jcb202211074 

 

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