Fluorescence Staining of Tubulin in Fiddler CrabsSam Boudeau’16 , Anna Schwartzberg - Bowdoin’17

Department of Biology, Colby College, Waterville, ME

Bioinformatics NCBI● Found lobster tubulin transcript and used as crustacean

reference ● Used ExPASy® to translate nucleotide sequence into amino

acid sequence● Used FASTA to align rabbit amino acid sequence to lobster

amino acid sequence● Determined 72% identity between rabbit and crustacean

(lobster) sequences, and concluded that commercially available rabbit tubulin antibody could be used

Introduction ● Circadian rhythms are endogenously-driven biological cycles with

periods of approximately 24 hours that drive biological processes and allow organisms to respond to environmental changes

● Our lab has found Melatonin, a hormone secreted at night, to be involved in the direct intracellular regulation of enzymes and free radicals in crustacean neurons

● Melatonin has neuroprotective and neuroregenerative effects in both mammalian and crustacean neurons (Figure 1)

Objectives● Investigate the effects of Melatonin on cytoskeletal organization in

cultured crustacean neurons using immunocytochemistry● Develop a protocol to efficiently quantify the growth observed in cultured

neuron using the software FIJI®

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Figure 3. Cells observed under a fluorescence microscope showing tubulin (green) outside the nucleus (magenta) of the stained cell

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Figure 2. Single FASTA alignment of crustacean and rabbit tubulin

Figure1. Cell 1 (untreated) shows significantly less growth than Cell 2 (treated with 3.6 μl of melatonin)

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Fiji® ● Developed a protocol to quantify neurite growth using FIJI® Image J

software with a Bamboo Tablet● Five Variables to Determine Growth:

a. Total Branch Lengthb. Number of Veilsc. Veil Aread. Gross Areae. Number of Growth Cones

Immunocytochemistry● Melatonin may directly or indirectly interact with tubulin, a

cytoskeletal protein, to increase neurite growth● Cultured crustacean neurons were stained for tubulin using

rabbit anti-tubulin as primary antibody, followed by Alexa Fluor 488 (green)

● Crustacean neuron nuclei were stained with DAPI (magenta)

Conclusions:● Cells treated with Melatonin were found to have more neurite

growth on average compared to cells that were not treated● Cells stained for tubulin showed the protein to be present

throughout the cytoskeleton with a higher concentration near membrane structures

References and AcknowledgementsThank you to Dr Andrea Tilden, Dr Andrew Christie, and The Colby Biology Department

Anger K, Dawirs RR, Harzsch S (1997) Immunocytochemical detection of acetylated alpha-tubulin and Drosophila synapsin in the embryonic crustacean nervous system. University of Bielefeld, Germany 41(3): 477-484. Barros C, Castro A, E Dupre, Perez C, Roco M, Schatten G (1991) Localization of microfilaments and a tubulin-like protein in crustacean (Rhynchocinetes typus) spermatozoon. Molecular Reproduction and Development 28(4): 373-379. Allodi S, Ammar D, Müller YM, Nazari EM (2013) On the brain of a crustacean: a morphological analysis of CaMKII expression and its relation to sensory and motor pathways. PLoS One 8(5): e64855. Cary GA, Cuttler AS, Duda KA, Kusema ET, Myers JA, Tilden AR (2012) Melatonin: neuritogenesis and neuroprotective effects in crustacean x-organ cells. Comp Biochem Physiol A Mol Integr Physiol 161(4): 355-60.

Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM0103423.

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