文本說明session1-02李卓然

1、Flagellar growth of Vibrio alginolyticus during cell divisionZhuoran Li (李卓然)Mentor: Fan Bai Peking University, Beijing, China1Bacterial flagella is pivotal for bacterial mobility and immunogenicity 21Indirect method to study the distribution of old flagella during cell division draw contradicting c

2、onclusionsBisset K A. Microbiology (1951)Road, C. B. J. gen. Microbiol. (1962)All or noneSharing 4A direct study onflagella dynamic during cell division5Vibrio alginolyticus with a single polar flagella is used in this studyGuttenplan, S. B., Shaw, S. & Kearns, D. B. Mol. Microbiol. (2013).6Two Hypo

3、thesis for the localization of new flagella Old poleOld poleOld poleOld poleOld poleOld poleNew poleNew flagella grow from the old poleNew flagella grow from the new pole7Static imaging suggest new flagella grow from the old pole8Static imaging suggest new flagella grow from the old pole9Real-time f

4、luorescent imaging of single cellindicate new flagella grow from the old pole(before cell division is completed)0min10min20min30min10(after cell division is completed)0min10min20min30min40minReal-time fluorescent imaging of single cellindicate new flagella grow from the old pole11Temporal feature of

5、 new flagella growth and its relationship with cell divisionTotal 82 events observed12Time Cell division completion Flagella filament elongation13Hypothesis for mechanism of new flagella growing from the old poleOld poleOld poleOld poleOld poleOld poleOld poleNew poleProtein gather at pole before ce

6、ll division completion14Temporal restrictionSpatial restrictionBasal body componentFliGAssembled in the early phase of flagellar assemblyUsing suicide vector and conjugation to label FliG with eGFPFliFFliGFliG15FliG assemble at the old pole before cell division is completed0min60min70min80min90min10

7、0min110minFliG occur at poleCell division completion16FliG assemble at the old pole after cell division is completedFliG occur at poleCell division completion0min20min40min60min80min100min17FliG occur at pole both before or after cell division completionTotal 72 events observed18Time Cell division c

8、ompletion FliG assembly at poleFlagella filament elongation19FlhF regulate flagella localization & numberPromoting expression of flagellar genes & flagellar assemblyTakekawa N, Kwon S, Nishioka N, et al. Journal of bacteriology, 201620FliFFlhFFliGFliGFlhFFlhFFlhFFlhF0min5minFlhF gather at the old po

9、le23FlhF always accumulate at old poles before cell division is completed0min15min30min45min60min75minFlhF occur at poleCell division completion22FlhF accumulate at old poles before growth of flagella0min20min40min60minFlhF occur at poleCell division completionFlagella growth23Time Cell division com

10、pletion FlhF accumulationFliG assembly at poleFlagella filament elongation24Remaining question: How do FlhF get to the pole?Hypothesis 1: diffusion and captureRemember that FlhF only accumulate at 1 pole before cell divisionLaloux, G. & Jacobs-Wagner, C. J. Cell Sci. 2014.25How do FlhF get to the po

11、le?Hypothesis 2: FlhF-GTP binding related enzyme up-regulate during divisionGTP-binding motif: might be required for FlhF polar localizationGreen, J. C. D. et al. J. Mol. Biol. 391, 679690 (2009).0min5min26Acknowledgements Prof. Fan Bai, Ziyi Zhao, Yu Huo and all the members in Bai Lab Prof. Michio

12、Homma and Dr. Seiji Kojima Prof. Chien-Jung Lo and Xiangyu ZhuangThis work is supported by National Students Training Program for Innovation. 27Thank you and e questions!31Backup slices32Vibrio alginolyticus with a single polar flagella is used in this studyGuttenplan, S. B., Shaw, S. & Kearns, D. B

13、. Mol. Microbiol. (2013).Dual flagellar system: Lateral flagella (Laf) Polar flagella (Pof)Laf-defective mutant: VI05 (Pof+ Laf-)Furuno M, Atsumi T, Yamada T, et al. Microbiology. (1997) 30Sample preparation for fluorescent microscopyActivationTransferDilutionLoading: load onto chamber by polylysine

14、Observation: Spinning Disk or Olympas (100X oil objective)Dying 1: NanoOrange (1:20)Dye 1 Dye 1 Dye 1 31Sample preparation for fluorescent microscopyActivationTransferDilutionObservation: Spinning Disk or Olympas (100X oil objective)Loading: load onto chamber by polylysineDying 2: FM4-64(1:200)Dye 2

15、 Dye 2 32Static imaging suggest new flagella grow from the old pole33Suicide vectorChromosome Chromosome Chromosome Method using suicide vector to label proteins on genomeTakekawa N, Kwon S, Nishioka N, et al. Journal of bacteriology, 201634Method using suicide vector to label proteins on genomeTakekawa N, Kwon S, Nishioka N, et al. Journal of bacteriology, 2016Suicide vectoreGFPdownstreamgeneXE.coliConjugationeGFP+Electroporation Select for suicideV.alginolyticus 35Basa