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, 19-22. , 2662-2667. Annu. Rev. Cell Annu. Rev. re-constituted re-constituted , 1273-1282. Nat. Rev. Genet Nat. Rev. 167 121 :ra29. , 13377-13382. , 601-606. . . 1 105 . . 43 106 . . in vitro J. J. Muscle Cell. Res. Motil J. Cell Biol J. Custom Custom Modules Sci. Signal Related Citations Related J. Cell Biol J. J. Clin. Invest J. Nat. Genet KIF7 Kinesin Motor Assay This Issue contains , 417-441. 29 . Proc. Natl. Acad. Sci. U.S.A Proc. Natl. Acad. ,331-344. , 377-383. Goetz Goetz S.C. and Anderson K.V. 2010. 11 al. 1987. H.S. et Paschal J. and 2012. Howard Friel C.T. 33 S., and Ohi R. 2013. C.E., Gayek Walczak Biol Dev. al. 2004. C.J. et Lawrence, Liem K.F. Jr., He M., Ocbina P.J.R., and Anderson K.V. 2009. al. 2009. et Cheung H.O. A. al. 2011. et Putoux et al. 2011. C. Dafinger 5. 6. 7. 8. 9. on available proteins and services, contact tservice@cy information. more for toskeleton.com Figure 1. KIF7(1-370) Motor Domain Activity. kit is directly The ELIPA Kinetic nm in the BK051/52 360 bance at absor into that the of the phosphate level proportional free assay is generated by enzymatichydrolysis of eitherATP orGTP. MT = microtubules. References 1. 2. 3. 4. Cytoskeleton’s Motor Werks Screening Services Screening Werks Motor Cytoskeleton’s At Cytoskeleton, we have developed assays fiedfor proteins 11 and recombinant Kinesin motor domains puri Kinesin families 8 of the 14 recognized represent that In addition, we have developed assays proteins and of cytoplasmic dynein and cardiac, skeletal, smooth, and non-muscle as myosins, well as an as a com available are These assays sarcomere. cardiac as well service with individual pound motors screening as multi-motor protein panel projects. Ifyou need a dif ferent motor protein or assay, we offer custom protein expression/purification and assaydevelopment services information more For forward. project your move help to ------. While 1 -expressed -expressed E. coli . Loss-of-function mu 2 . Additionally, other of members the . Additionally, 6 , , which are processes that are expected 8 or Domain Exhibits Microtubule-Stimu or MT depolymerization (Kinesin-8 and -13 . Given the important role of Hh signaling in signaling Hh of role important the Given . 7 3,4 www.cytoskeleton.com nucleotide preference of the KIF7 motor domain motor KIF7 the of preference nucleotide , it will be interesting to see if research in this area area this in research if see to interesting be will it , 5 . Mice lacking KIF7 exhibit a similar phenotype to mice to a similar phenotype . Mice lacking KIF7 exhibit 1 for its for motor activity to shown been have proteins of motor superfamily Kinesin modulate tubulin polymerization (Kinesin-7 and -10fam ily members) members) family activity. GTPase of tubulin the level affect to nesins are typically characterized as having MT-dependent MT-dependent as having characterized typically are nesins either suggest a these data Consequently, activity. ATPase fide bona for GTP or over perhaps ATP, the GTPase activity is actu is that in a manner MTs tubulin in the from ally coming heavy the kinesin that KIF7. It is noteworthy by enhanced ATP is able GTP to asutilize well as chain, Kinesin-1 family, noise ratios. Recently, we have developed an assay for KIF7 for assay an developed have we Recently, ratios. noise and expressed using the recombinantly family) (Kinesin-4 purifiedKIF7(1-370) motor domain. This with our ATPase/ and when tested is >80% pure protein a we discovered BK051/52), Kit (Cat# ELIPA Kinetic GTPase very strong microtubule (MT)-dependent GTPase activity (Fig. 1). This was a very unexpected finding given that ki cancers also uncovers a role for KIF7 in Hh signaling-dependent cancers. The Kif7 Mot Activity GTPase lated continuesCytoskeleton to expand their offerings of kine sin, dynein, and myosin motor assays with high signal to brain is located outside the skull) outside the is located brain tations in theKIF7 gene in humansresults in ciliopathies abnormalities (e.g., developmental lethal lead to can that less or syndrome) hydrolethalus fetal and Acrocallosal severe developmental defects (e.g., Meckel and Joubert syndromes) (BCC) and other of basal cell carcinomas development the tively regulates GLI transcription factor activity, the presence and in of Hh signaling it positively influences GLI dependent signaling; possibly through KIF7’s Hh-depen dent translocation from the base of primary ciliato the tip lacking the GLI3 transcriptionfactor, which is character (i.e., and exencephaly toes) (i.e., extra polydactyly by ized to play an important role in embryonic development and development in embryonic role an important play to of basal cell carcino development in the a role play may mas. KIF7 functions in Hedgehog (Hh) signaling through the negative and positive regulation of the GLI family of transcription factors (i.e., GLI1, GLI2, and GLI3) the mechanism of this regulation isstill being elucidated, it appears that in the absence of an Hh ligand, KIF7 nega A Snapshot of KIF7 in Disease of KIF7 in A Snapshot shown has been member that family is a Kinesin-4 KIF7

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sarcomere Myosin and the January 2014 clinical targets clinical Molecular motors as Molecular motors December 2013 citations Tubulin screening Tubulin November November 2013 Future Topics Future

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Our recently expanded Custom Service department provides additional resources for your research projects. Cytoskeleton is leading the way to develop novel kinesin, dynein, and myosin based compound screens. We are scientists dedicated to providing accurate data reported in a detailed and timely manner.

About Custom Services Clients Include: Like our regular product offerings, the Custom from more than twenty defined modules • Merck & Co., Inc. • Astra-Zeneca plc Service department emphasizes quality (for a full list, visit www.cytoskeleton. • Eli Lilly & Co. • GlaxoSmithKline plc products and services. We understand com/custom-services), and then contact • Amgen, Inc. • Genentech, Inc. that accuracy and timeliness are critical Technical Support (tservice@cytoskeleton. • Abbott Laboratories • Johnson & Johnson elements for a successful project. Choose com) to guide you through the process. • Pfizer, Inc. • Bristol-Meyers Squibb

Let’s get started, it’s as easy as 1,2,3 ... 1. Choose a module and ask for a quote (24h turn around time) 2. Review quote, specifications, and deliverables 3. Place order and receive regular updates until project is finished Compound Screening Modules

Type Format Deliverable Module # Timeline (wks) Microtubule stimulated ATPase assay, 96 assays, consisting of 40 duplicate single concentrations (or 5 x CDS050 or 2 Eg5 Kinesin motor assay kinetic, absorbance at 360nm IC50s), plus eight control wells. PDF Report with Executive Summary, CDS051 Introduction, Methods, Results and Data Analysis. Ca2+/Sarcomere (thin filament) CDS056 2 Cardiac Myosin motor assay stimulated ATPase assay, kinetic, Same as CDS052. absorbance at 360nm Microtubule stimulated ATPase assay, CDS065 2 Dynein motor assay kinetic, absorbance at 360nm Same as CDS052.

Tubulin (>99% pure) Polymerization 96 assays, with 40 duplicate single concentrations or 5 x IC50s, plus CDS009 or 2 Tubulin polymerization Assay, kinetic, fluorescence at eight control wells (vinblastine, nocodazole or taxol). PDF Report with CDS010 360nm/410nm Executive Summary, Introduction, Methods, Results and Data Analysis. GTP exchange factor plus Small 60 assays consisting of either 28 duplicate reactions plus 4 controls, CDS100 2 G-protein (e.g. Rho or Ras) with mant- or 5 x IC50s plus 1 x control IC50. PDF report with Executive Summary, GEF/GTPase exchange assay GTP reporter. Kinetic, fluorescence at Introduction, Methods, Results and Data Analysis. 360nm/450nm

Gene Cloning and Protein Purification Modules

Type Name Deliverable Module # Timeline (wks) Recombinant Small Protein Small protein or protein Highly purified, His-tagged active protein lyophilized in 10 x 100 µg aliquots REC012 3 domain (<30 kDa) with gene (or more depending on yield). Datasheet and assay method. Activity in line provided by client with published articles. E. coli expression. Recombinant Small Protein Small protein or protein Same as above with gene synthesis. REC022 6 plus cloning domain (<30 kDa) including gene synthesis Recombinant Kinesin Motor- Medium to large protein Same as REC012. REC032 3 Protein or protein domain (30-100 kDa) Recombinant Kinesin Motor edium to large protein or Same as above with gene synthesis. REC042 8 Protein plus gene cloning protein domain (30-100 kDa) with gene synthesis Native or eukaryotic protein Cited protein purification Same as above plus using a published procedure. REC052 4-20 expression & purification

Assay Development Modules 5500 Cdc42 + Dbs

5000 Type Name Deliverable Module # Timeline (wks)

4500 GTP Exchange G-protein GTP exchange Report with optimized protocol, based on data from titrating four variables DEV026 4 4000 (fluor. kinetic, 360nm/460nm) assay using Mant-GTP ([ionic], [MgCl2], [Mant-GTP] and temp.). 3500 GTPase assay GTP hydrolysis assay, Same as above, except [Mant-GTP] is replaced by [G-protein] and if avail- DEV031 4 Cdc42 only 3000 (abs, endpoint, 650nm) detecting phosphate able [GAP protein].

2500 Nucleotide exchange (RFU) 0 300 600 900 1200 1500 1800 Motor ATPase ATP hydrolysis assay, Report with optimized protocol, based on data from titrating five variables DEV034 4 Time (s) (abs, kinetic, 360nm) detecting phosphate ([ionic], [MgCl2], [Motor], [microtubules] and temp.).

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