FNA_200814_Neo_GatastatinG2

403 人阅读发布时间：2020-09-15 11:26

Background

γ-tubulin?

Amongthememberofthetubulinfamily,α-,β-,andγ-tubulinsareubiquitouslyexpressed.
α-andβ-tubulinsformheterodimers(α/β-tubulin)andpolymerizedtomicrotubules(MTs).
Ontheotherhand,γ-tubulinisnotacomponentofMTsbutplaysaroletoformγ-tubulinringcomplex
(γTuRC).γTuRCplaysanimportantroleasaninitiationpointofMTpolymerizationfromcentrosome.
However,themolecularmechanismofγ-tubulin-basedMTinitiationandregulationisstillunclear.

Gatastatin?

Gatastatinwasoriginallydiscoveredin2015astheworld’sfirstγ-tubulin-specificinhibitor.
In2020,asuperiorderivativeofGatastatin,calledsecondgenerationGatastatin(GatastatinG2)
wasidentifiedbyDr.UsuigroupatUniversityofTsukuba.
GatastatinG2showsaboutten-foldshigherinhibitionactivitythanGatastatinincell-basedexperimentsbuthaslittleeffectsonα/β-tubulinpolymerization.

Features
● Specific inhibitor for γ-tubulin.
● Little effect on α/β-tubulin polymerization.
● Block nucleation of microtubule from centrosome.
● Induces abnormal chromosome alignment and multipolar formation in mitotic cells.
● Useful for functional analysis of γ-tubulin in mitosis.
● Immediate inhibitory effect compared with gene regulation approach, such as RNAi.

Specificity
EffectofGatastatinG2onMTpolymerizationinvitro
Purified tubulin (1 mg/ml) from porcine brain was polymerized by 0.8 M glutamate for 30 min at 37oCwith or without 10 μM colchicine (an inhibitor of MT), 10 μM paclitaxel (an activator of MT polymerization) and 30 μM GatastatinG2. Polymerized and unpolymerizedtubulins were separated by ultracentrifugation. Polymerization ratio (%) was estimated by SDS-PAGE. Colchicine clearly inhibited MT polymerization, paclitaxel promoted MT polymerization but GatastatinG2 showed little effect on MT polymerization.

Application Data
GatastatinG2 induced abnormal spindle formation in mitotic cells
GatastatinG2 treated HeLa cells were fixed and stained with anti-α-tubulin (spindle fibers), anti-pericentrin(centrosome) and DAPI (chromosomal DNAs). The resulting spindle morphology was classified and quantified. At the lower concentration of GatastatinG2, misaligned chromosomes were mainly observed. On the other hand, high concentration often induced multipolar spindle formation rather than congressionerror. Multipolar induction was clearly dose-dependently observed. This experiment suggests γ-tubulin regulates both chromosome movement and normal bipolar formation in mitotic cells.
Gatastatin G2 blocks centrosome-derived MT formation in mitotic cells
HeLa cellswere cultured and subsequently treated with S-trityl-L-cysteine (STLC) Then, the cells were washed with ice-cold medium and incubated on ice to depolymerized MT. The cells were treated with 1% DMSO, or 0.03-3 μMGatastatin G2 for 15 min on ice. After drug treatment, the cell media was exchanged with warm (30oC) media containing drugs and the cells were further cultured at 30oCfor 3 min. The cells were fixed and stained with anti-α-tubulin and anti-pericentrinfor centrosomes. GatastatinG2 clearly inhibited MT initiation from centrosomes dose-dependently in mitotic cells.

Product Information [ Manufacturer : FNA ]
Product Name	Code	Size	Storage
GatastatinG2 <γ-Tubulin Inhibitor>	FDV-0040	0.1 mg	-20°C

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