4.Dynamics(4.Dynamics( 动态动态 ) of YFP-SCAMP–Labeled Organell) of YFP-SCAMP–Labeled Organelles in the Cytoplasmes in the Cytoplasm

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

Result: the organelles labeled by SCAMP1-YFResult: the organelles labeled by SCAMP1-YFP may represent endosomal compartments in BP may represent endosomal compartments in BY-2 cellsY-2 cells

5.YFP-SCAMP1–Labeled Cytoplasmic Organelles Are 5.YFP-SCAMP1–Labeled Cytoplasmic Organelles Are Distinct from the Golgi and the PVCDistinct from the Golgi and the PVC

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

Conversely, the drug wortmanninConversely, the drug wortmannin （青霉素） （青霉素） wawas without effect on the Golgi , but caused the PVCs.s without effect on the Golgi , but caused the PVCs.

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

First,we want to know whether the labelFirst,we want to know whether the labeled ed YFP-SCAMP1 or SCAMP1-YFP belonged YFP-SCAMP1 or SCAMP1-YFP belonged to PVC. So we use the BFA.to PVC. So we use the BFA.

Golgi marker Gonsti-YFP Golgi marker Gonsti-YFP different concentrationdifferent concentration

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

Then,is it labeled in Golgi?Then,is it labeled in Golgi?we use wortmannin we use wortmannin

GolgiGolgi without effect without effect

not not GolgiGolgi

Result:Result: SCAMP-YFP or YFP-SCAMP-labeled strcturSCAMP-YFP or YFP-SCAMP-labeled strctur

es did not behave as Golgi or PVC in transgenes did not behave as Golgi or PVC in transgenic BY-2 cellsic BY-2 cells

6. YFP-SCAMP1–Labeled Organelles 6. YFP-SCAMP1–Labeled Organelles Are Early EndosomesAre Early Endosomes

cis-cis-Golgi marker Man1-GFP ,Golgi marker Man1-GFP ,the the trans-trans-Golgi marker GONST1-YFP .Golgi marker GONST1-YFP .

the PVC marker GFP-BP-80the PVC marker GFP-BP-80 not overlapnot overlap

Similarly, organelles labeled by SCAMP1a antSimilarly, organelles labeled by SCAMP1a antibodies were also different from Golgi or PVC ibodies were also different from Golgi or PVC markers in transgenic BY-2 cells .markers in transgenic BY-2 cells .

These results again show that the SCAMP1-poThese results again show that the SCAMP1-positive organelles were not Golgi or PVC. sitive organelles were not Golgi or PVC.

FM4-64, a fluorescent styryl dye(FM4-64, a fluorescent styryl dye( 染料染料 ) , as a mark) , as a marker for the endocytic pathway(er for the endocytic pathway( 内吞途径内吞途径 ) in plant ce) in plant cells .lls .

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

GolgiGolgi

PVCPVC

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

SCAMP1-YFP–labeled organelles represent eiSCAMP1-YFP–labeled organelles represent either an early endosomal (ther an early endosomal ( 初级内体初级内体 )compart)compartment or a recycling endosome in BY-2 cellsment or a recycling endosome in BY-2 cells

However,this claim is based on an indirect comparisonHowever,this claim is based on an indirect comparison

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

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

VSR-marked PVCsVSR-marked PVCs

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

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

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