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