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TOWARDS UNDERSTANDING THE IMPORTANCE OF CENTRAL PAIR APPARATUS PROTEINS IN THE FLAGELLA OF UNICELLULAR
ALGA, Chlamydomonas reinhardtii
SUBMITTED BYURJA ACHAL BHATT
BBT-1-11010
UNDER THE GUIDANCE OF DR. JACINTA D’SOUZA
UM-DAE-CBSKalina Campus, Santacruz, Mumbai
Introduction
Chlamydomonas reinhardtii species
Flagella- Structure and Organization
Axoneme
Central Pair Apparatus Proteins
Cell Architecture
FlagellaAxoneme
CP proteins with Adenylate kinase domain
Wild type strains- CC124,CC125 and CC3395
Phenotype- two anterior flagella
Presence of all projections
Cpc1 strain
Phenotype- flagella with reduced beat frequency
C1b projection absent
Hydin mutant
Phenotype- hands-up hands-down
C2b projection absent
Pf-18 strainCPC absent complete paralysis
Hydin; cpc1 mutantC1b, C2b absentComplete paralysis
MethodologyGeneration of resources
Transformation of RNAi constructs into wild type and cpc1 mutants
Screening of mutantsVisual screening- using microscopyRNA extractioncDNA synthesis and transcript analysis of hydin
Reactivation Assay
8kb (shown as red arrows) pKL3hyAS plasmid linearized with Ecor1 as well as Dra 1
Construct provided by Prof. Lechtreck (Univesity of Georgia)
Selectable marker for arginine auxotroph CC3395. 8 kb ARG 7 construct obtained by double digestion using Hind III and Xba I.
pChlamiRNA2 plasmid
Template for PCR
Amplification of aphVIII gene cassette – paromomycin resistance
A. All the 8 lanes (replicates of the PCR reaction) mixture showed successful amplification of the 1.8 kb aphVIII gene using the high-fidelity KOD-FX Polymerase at 55.6°C annealing temperature.
A. Arrows indicate the amplicon of 1.8 kb aphVIII gene. 55.6°C chosen as the optimum temperature.
Amplification of aphVIII gene cassette – paromomycin resistance
Autolysin
Add Incubate for hour
Check for autolysin
action efficiency
Resuspend pellet in fresh TAP
Cells without
Cell wall
Glass bead eppendorf
PEGSelectable Marker
pKL3hyAS
Remove transformed cells
Wash the beads with sterile TAP
Resuspend cells in 10 mL sterile TAP
Incubate for 16 hrs at 25°C
Resuspend pellet in TAP
Plate the cells on TAP plates with appropriate selectable marker
Incubate plates for obtaining transformed colonies
Strain To obtain Template DNA
Selectable Marker
Antibiotic/Supplements to the medium No. of colonies
CC124 Hydin mutant pKL3hyAS aphVIII Paromomycin
81- EcoR181-Dra1
CC3395Hydin
mutant pKL3hyASARG7 Arginine 38
aphVIII Paromomycin Results awaited
Cpc1Hydin; cpc1
double mutant
pKL3hyAS aphVIII Paromomycin4-EcoR1
5-Dra1
A. Representative plates showing transformants.
B. Experimental controls
A
B
SCREENING
cDNA synthesis followed by transcript analysis using hydin specific primers.
RNA was extracted for 11 prospective double mutants.
Cells contained in every well were checked for peculiar phenotype using bright-field compound microscope.
Inoculation of transformants from patched plates in 96 well ELISA plate.
cDNA synthesized using OligoDT
cDNA synthesized using Random Hexamers
140 bp actin and 220bp hydin band observed
140bp Actin gene amplified
Spurious 90bp band obtained for hydin
A 220bp hydin amplicon was optimum at annealing temperatures of 57 °C, 55.7 °C & 55 °C
1A11 1A8 1A9 1B3 1B5 CPC1
Ladder H A H A H A H A H A Ladder A H
PCR of Prospective Double Mutants and cpc1 with Hydin and Actin primers
•Hydin and actin expression level in the prospective double mutants and their comparison with cpc1. •The arrows represent 220 bp hydin PCR product and the stars represent 140 bp actin PCR product.1B3 was thought to be cpc1;hydin double mutant.
• Due to the instability of the RNAi in Chlamydomonas line, the immotile cells reverted back to wild type
phenotype and currently efforts are undergoing in the lab to produce stable RNAi lines of Hydin
Reactivation Assay
Washing
• HES buffer• Cells are pelleted and washed thrice
De-membran
ation
• HMDEKP buffer+IGEPAL• Flagellar membrane solubilized
Reactivation
• HMDEKP buffer+500µM ATP• Cells turn motile
Reactivation Assay with CC124De-membranated CC124 cells
Reactivated CC124 cells
Reactivation Assay with cpc1De-membranated cpc1 cells
Reactivated cpc1 cells
Graphical Representation of Relative Speed of CC124 and cpc1
•Ten-fold decrease in relative speed of cpc1 was observed as compared to wild-type due to the absence of C1b projection of the CP apparatus.
•cpc1 mutant even after reactivation cannot achieve a speed as optimal as the wild type cells, indicating the requirement of these proteins for the purpose of motility
Conclusion• Chlamydomonas serves as a good model to
study motility• Central pair proteins perform essential
function which contribute towards motility• RNAi is a good tool, however due to
instability, an alternative approach needs to be adopted like insertional mutagenesis.
• Reactivation of Chlamydomonas cells is a very good assay to differentiate whether proteins knocked down only generate ATP or also perform structural roles.
Conclusion
• The study thus provided a ready-reckoner for efficiently obtaining, screening and testing the single and double mutants for hydin knockdown and subsequently studies the role of adenylate kinases in the central pair