PARKHURST LAB -- Reagents and Protocols
Bnr8_Fridge2

Reagents and Protocols


Parkhurst Lab reagents available from the Development Studies Hybridoma Bank

Antigen monoclonal line species refs
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p120catenin P1B2 fly 3, unpublished
p120catenin P4B2 fly 3, unpublished
dSir2 P2E2 fly 4, unpublished
dSir2 P4A10 fly 4, unpublished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

  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)
28285 w*; wash[Δ185]/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  (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

• 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

• 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

• Pak3 (GFP fusion protein under control of the UASp promoter (inducible).
52302
w[*]; P{w[+mC]=UASp-mChFP-Pak3}30 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 DrosophilaGenes 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.

Protocols