Reagents

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Parkhurst Lab reagents available from the Development Studies Hybridoma Bank

Antigen Monoclonal Line Species Reference
Rho1 P1D9 fly 3
Wash P3H3 fly 8, 12
Wash P4C9 fly 12
WASP P5E1 fly 12
WASP P3B1 fly 12
SCAR P1C1 fly 12
SCAR P1C8 fly 12
Whamy P4A8 fly 12
Whamy P1D1 fly 12
p120catenin P1B2 fly 3, unpublished
p120catenin P4B2 fly 3, unpublished
dSir2 P2E2 fly 4, unpublished
dSir2 P4A10 fly 4, unpublished

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

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

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}     3nd 11

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 3nd 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

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

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 Dev5, 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 Biol8, 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 Biol193, 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. Dyn241, 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)