□ 형광 단백질을 이용한 살아 있는 세포내 actin 모니터링

● Live-cell monitoring of actin dynamics

● Easy to use, dual color assay

이 제품은 살아있는 세포에 있는 actin filament 시스템의 역동적인 변화를 모니터링 하기 위해 설계되었다. 이 제품에는 DD-AcGFP1 (green, destabilized) 과 mCherry (red)에 융합하는 actin을 코딩하는 lentiviral vector와 DD를 안정화시키는 리간드 Shield1를 포함하고 있다.세포 배양시 리간드 Shield1를 넣지 않으면 세포내 DD-AcGFP1-Actin은 proteasome에 의해 분해된다. 반면, DD를 포함하지 않은 mCherry-Actin은 발현되어 세포내에 존재한다.Shield1의 첨가와 제거를 통해 pulse-chase와 같은 조건을 셋팅할 수 있다. 이것을 통해 기존의 (red) mCherry-Actin actin filament network로 Shield1(green)에 의해 안정화된 새로운 DD-AcGFP1-Actin가 껴들어가는 actin monomer의 중합화(polymerization)를 모니터 할 수 있다.Observe actin filament remodeling using the Lenti-X Actin Dynamics Monitoring Kit. Actin filaments are completely remodeled in HeLa cells in less than 1 hr. Panel A. Self-assembly of actin filaments occurs at the plus end of an existing actin filament as monomeric actin is incorporated. Conversely, disassembly occurs at the minus end where actin monomers depolymerize from the filament, causing a continuous rearrangement of the actin filament network. HeLa cells were infected with constructs encoding mCherry human alpha-Actin and DD-AcGPF1 human alpha-Actin. Cells were fixed using 4% paraformaldehyde and imaged with a 40X objective. Fluorescence micrographs were taken 1 hr after addition of Shield1 (Panels E-G) or without Shield1 (Panels B-D). In the absence of Shield1, DD-AcGFP1-Actin was degraded quickly (Panels C & D) despite a normal, mCherry-labeled actin filament network (Panels B & D). In the presence of Shield1, DD-AcGFP1-Actin was stabilized and present in the actin filament network along with mCherry-labeled Actin (Panels E-G). Our results are in agreement with a previous report that the actin filament network rearranges completely in 1 hr in PtK2 epithelial cells, but we used the extremely simple ProteoTuner-based method, rather than the extremely laborious microinjection method.