4 dynamics of yfp scamp labeled organelles in the cytoplasm
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4.Dynamics( 动态 ) of YFP-SCAMP–Labeled Organelles in the Cytoplasm. The cytoplasmic organelles labeled by the SCAMP1-YFP or YFP-SCAMP1 were highly mobile. they moved to, moved along, and moved away from the plasma membrane(PM) in transgenic BY-2 cells.

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4 dynamics of yfp scamp labeled organelles in the cytoplasm
4.Dynamics(动态) of YFP-SCAMP–Labeled Organelles in the Cytoplasm

  • The cytoplasmic organelles labeled by the SCAMP1-YFP or YFP-SCAMP1 were highly mobile. they moved to, moved along, and moved away from the plasma membrane(PM) in transgenic BY-2 cells.



5 yfp scamp1 labeled cytoplasmic organelles are distinct from the golgi and the pvc
5.YFP-SCAMP1–Labeled Cytoplasmic Organelles Are Distinct from the Golgi and the PVC

  • We know:the drug BFA (brefeldin A) at low concentrations (5 to 10 μg/mL) caused the aggregation(聚集) of Golgi stacks(垛叠) in transgenic BY-2 cells but PVCs remained unaffected.

  • Conversely, the drug wortmannin(青霉素) was without effect on the Golgi , but caused the PVCs.

  • so, these differential drug treatments serve as reliable tools to identify and distinguish the Golgi from the PVC in transgenic BY-2 cells.


4 dynamics of yfp scamp labeled organelles in the cytoplasm

  • First,we want to know whether the labeled from the Golgi and the PVCYFP-SCAMP1 or SCAMP1-YFP belonged to PVC. So we use the BFA.

  • Golgi marker Gonsti-YFP different concentration

  • PVC marker GFP-BP-80aggregation --not pvc


Then is it labeled in golgi we use wortmannin golgi without effect not golgi
Then,is it labeled in Golgi? from the Golgi and the PVCwe use wortmannin Golgi without effectnot Golgi


4 dynamics of yfp scamp labeled organelles in the cytoplasm

  • Result: from the Golgi and the PVC

  • SCAMP-YFP or YFP-SCAMP-labeled strctures did not behave as Golgi or PVC in transgenic BY-2 cells



Cis golgi marker man1 gfp the trans golgi marker gonst1 yfp the pvc marker gfp bp 80
cis- from the Golgi and the PVCGolgi marker Man1-GFP ,the trans-Golgi marker GONST1-YFP .the PVC marker GFP-BP-80

not overlap


4 dynamics of yfp scamp labeled organelles in the cytoplasm


4 dynamics of yfp scamp labeled organelles in the cytoplasm

  • FM4-64, a fluorescent styryl dye( also different from Golgi or PVC markers in transgenic BY-2 cells .染料) , as a marker for the endocytic pathway(内吞途径) in plant cells .

  • make sure whether the YFP-SCAMP1 or SCAMP1-YFP–labeled organelles were on the endocytic pathway, we performed uptake studies with FM4-64 on transgenic BY-2 cells expressing different reporters.


Golgi
Golgi also different from Golgi or PVC markers in transgenic BY-2 cells .

PVC


4 dynamics of yfp scamp labeled organelles in the cytoplasm

  • Therefore, we sure that the FM4-64 reached the SCAMP1-YFP–labeled compartments before the GFP-BP-80–labeled PVCs.

  • SCAMP1-YFP–labeled organelles represent either an early endosomal (初级内体)compartment or a recycling endosome in BY-2 cells


4 dynamics of yfp scamp labeled organelles in the cytoplasm

  • However,this claim is based on an indirect comparison SCAMP1-YFP–labeled compartments before the GFP-BP-80–labeled PVCs.

  • In addition, it’s also possible that overexpression(过表达) of SCAMP1-YFP may lead to grow of FM4-64 uptake in cells expressing SCAMP1-YFP compared with BY-2 cells expressing the PVC marker.

  • so we studies on wild-type (野生型)BY-2 cells using the fixable form of the styryl dye, AM4-64, to compare the time course of dye arrival into VSR-labeled PVCs against SCAMP-labeled organelles.


4 dynamics of yfp scamp labeled organelles in the cytoplasm

  • VSR-marked PVCs SCAMP1-YFP–labeled compartments before the GFP-BP-80–labeled PVCs.


4 dynamics of yfp scamp labeled organelles in the cytoplasm

  • Therefore, the organelles labeled by either SCAMP1-YFP or YFP-SCAMP1 must represent an endocytic compartment upstream(上游) from the PVCs.

  • So,SCAMP1-labeled organelles as early endosomes in BY-2 cells.

  • However, because AM4-64 did not localize with SCAMP1-labeled organelles during the very early stages of uptake, these AM4-64–labeled organelles may also represent a different population of early endosomes distinct from those labeled by SCAMP1.