Light alters the expression Light alters the expression of of -tubulin in octopus retinas-tubulin in octopus retinas

Sachin Singh, Gina H. Ochoa and Laura J. Robles, Department of Biology

California State University, Dominguez Hills, Carson, CA

IntroductionIntroduction

• Our lab is trying to determine signaling pathways that regulate cytoskeletal organization in photoreceptor cells of the octopus retina.

• Dramatic changes in photopigment distribution and rhabdom morphology have been reported in light/dark-adapted octopus retinas (Torres et al., 1997, Cell Tissue 290:167). These changes may be attributed to the reorganization of tubulin and actin based cytoskeletons.

• Light regulates tubulin mRNA expression in plants.

• Immunocytochemical and in situ RT-PCR experiments from our lab show that tubulin is distributed differently in the light and dark

Octopus bimaculoidesOctopus bimaculoides Retinal Tissue Retinal Tissue

Rhabdoms

Pigment layer

Inner Segment

Longitudinal section of retina tissue Longitudinal Diagram Cross Section

Rhabdoms

Plexiform layer

Magnification= 20X

Dr. Ying Mei ClarkDr. Ying Mei Clark

Immunofluorescence

Light-adapted, ventral and dorsal halves

Hassan Elfarissi – M.A. thesisHassan Elfarissi – M.A. thesis

ImmunofluorescenceDark-adapted, ventral and dorsal halvesDark-adapted, ventral and dorsal halves

Hassan Elfarissi – M.A. thesisHassan Elfarissi – M.A. thesis

HypothesisHypothesis

• Because tubulin mRNA and protein are distributed differently in light- and dark-adapted retinas, we will test the hypothesis that light regulates the expression of tubulin mRNA.

-We will measure the amount of -tubulin mRNA expressed in the light and dark to determine if there are quantitative differences.

-Furthermore, we believe that the expression is also different in the dorsal and ventral halves of the

retina.

MethodsMethods

• We are using Northern blot analysis to test our hypothesis. – Animals: Octopuses were LA and DA for 3-4 hours.– mRNA isolation: Retinas were homogenized, mRNA was

isolated (Ambion Poly(A) RNA kit).– DIG -tubulin probe was prepared using PCR methods. – Northern blot analysis: First, mRNA separated on

formaldehyde-agarose gels, transferred and hybridized with an DIG -tubulin probe. This method was used with DA dorsal, DA ventral, LA dorsal and LA ventral halves of the retina.

– Results were quantified using a Bio-Rad Versadoc 3000.

ResultsResults

Blot 1 Blot 2

Marker LA DA

-tubulin size:1.5-1.8 kilo base pairs

ResultsResults

DA LA DD DV Video print

Blot 3

-Quanitation Results

ConclusionsConclusions

• Northern blots confirmed that the expression of -tubulin mRNA is different between light- and dark-adapted retinas.

• The results show that there is more - tubulin expression in the dark than in those retinas maintained in the light.

• Furthermore, we detected no difference in the expression of -tubulin mRNA between dorsal and ventral halves of retinas within their respective lighting conditions.

Conclusions (cont.)Conclusions (cont.)

• Alpha-tubulin cytoskeletal remodeling may be involved in the structural changes that occur in the octopus retina during light- and dark-adaptation.

• Other results show that the small GTPase Rac may be involved in the signaling pathway, regulated by light, that leads to changes in expression of tubulin mRNA.

• These results are being further tested using real time RT-PCR.

Further ResearchFurther Research

• Running Northern blot analysis with positive controls.

• Furthermore, I will do northern analysis with -actin housekeeping gene to check the results of previous blots.

AcknowledgementsAcknowledgements

The authors would like to thank the following people for their contribution:

– Josephine Mendoza

– Freddie Zuniga

– Ying Mei Clark

– Teresa Ramirez

– Hassan Elfarissi

– Tigress Briggs

– Special thanks to Clementine Sessoms for urging me

to present.Supported by NIH NIGMS

Minority Biomedical Research SupportResearch Initiative for Scientific Enhancement (RISE)

GM62252Support of Continuous Research Excellence (SCORE)

GM08156