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

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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.
slide4
First,we want to know whether the labeled YFP-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
slide6
Result:
  • SCAMP-YFP or YFP-SCAMP-labeled strctures did not behave as Golgi or PVC in transgenic BY-2 cells
slide9
Similarly, organelles labeled by SCAMP1a antibodies were also different from Golgi or PVC markers in transgenic BY-2 cells .
  • These results again show that the SCAMP1-positive organelles were not Golgi or PVC.
slide10
FM4-64, a fluorescent styryl dye(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.
slide12
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
slide13
However,this claim is based on an indirect comparison
  • 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.
slide15
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.
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