The Effects of Octanol on Gap Junctions and Actin of Neoblasts during Smed Planarian Regeneration

Ian Crosby, Marianne Gadiano, Shana Gregory, & Nkemdilim NdubuizuBIOL 340L Developmental Biology Laboratory, Spring 2016

University of Maryland, Baltimore County

AbstractPlanaria have an amazing ability to regenerate into entirely new

beings from 1/279th of a piece. Their ability to regenerate is due to their stem cells called neoblasts being able to proliferate and move to the site of damage before differentiating. Cell movement is mediated by cytoskeletal rearrangement, specifically, actin and myosin dynamics. This detailed process must be coordinated by signaling between cells. One way that cells can relay messages for coordinated movements is through gap junctions. However, if the signal is not relayed between cells or relayed improperly, cell migration is abnormal. We believe that by inhibiting the function of gap junctions using octanol, we will impair the signal for cell movement between cells, therefore altering the levels of actin and myosin in planarian neoblasts. Our data suggests that there is little correlation between gap junction inhibition and levels of actin within regenerating planaria. Understanding the signaling mechanism in planarian regeneration is important because it can be applied to the field of regenerative medicine.

Introduction❖ The remarkable regenerative abilities of the Schmidtea

mediterranea planarian have captured the attention of scientists who hope to apply this ability to humans, such as the regeneration of insulin cells for those with diabetes and the regeneration of nerve cells for those with spinal cord injuries.

❖ Following injury, the planarian cells begin the process of regeneration by contracting around the injury site, reducing the wound size. After 24 hours, local cell death peaks and neoblasts proliferate around the site [2]. Within 6-8 hours following injury, mitotic activity of neoblasts is at its peak. This peak is more sustained three days after injury. At these timepoints, it is thought that progenitors which were generated during wounding migrate to the injury site and form a regeneration blastema [4]. Within a week, the planarian would have regenerated to its full size.

❖ The process of regeneration would not be possible without sufficient cell communication. We believe that gap junctions, a specialized intercellular connection used to diffuse certain molecules directly between the cytoplasm of two different cells, and actin, the motility protein that is involved with localization of cells, may play a role in neoblast migration during planarian regeneration [3][1].

Experimental Design

Data/Results

References[1] A Mogilner et al(1996). Call motility driven by actin polymerization. Biophys J. 71(6): 3030–3045.[2] Harshani, P. T., Hoyer, K. K., & Oviedo, N. J. (2014). Innate immune system and tissue regeneration in planarians: An area ripe for exploration. Seminars in Immunology, 26(4), 295-302.[3] N.J. Oviedo et al (2010) Long-range neural and gap junction protein-mediated cues control polarity during planarian regeneration. Dev. Biol., 339 , pp. 188–199[4] Rink, J. (n.d). Stem cell systems and regeneration in planaria. Development Genes And Evolution, 223(1-2), 67-84.

Discussion❖ Under the microscope, it seems that regeneration and

movement was not hindered with the increasing concentrations of octanol for all three timepoints. (Figs. 2-4)

❖ Looking at the data from the western blots, it appears that the levels of actin remained fairly constant. (Figs. 5-7) However, this would need to be determined using a loading control.

❖ Actin band was vivid in the 18-hr 0% octanol planaria heads (Figure 8).

❖ Most actin bands were too faint to tell whether actin levels have changed while the planaria were exposed to octanol. (Figs. 8-10) This may be due to sample evaporation during PCR or insufficient loading of samples onto the gels.

❖ Based on how far the actin bands travelled, it seems that actin is consistent at all concentrations and timepoints.

❖ It seems that actin and gap junctions have little correlation. More experiments will need to be conducted.

Future Directions❖ Gap junctions might regulate the certain secondary

structures of actin which could lead to the janus heads observed when planaria are treated with octanol, and so we would like to repeat the experiment focusing on these structures.

❖ Examine presence of filamentous actin through the use of immuno fluorescence.

Figure 1: Well plate were set-up with different octanol concentrations and timepoints; H = heads, T = tails, 0 = planaria water (negative control), 25 = 25% concentration (63.5 uM), 50 = 50% concentration (127 uM), 75 = 75% concentration (190.5 uM).

Figures 2-4 (above, from left to right): 18 hour, 4 day, and 1 week planaria (respectively) in well-plate before dispensing in RNAlater.

KEY

Lane:1,10 Ladder

Lane:2,6 Negative Control

Lane:3,7 25%

Lane:4,8 50%

Lane:5,9 75%

Figure 8 (Top Left): 18 hour PCR gelFigure 9 (Bottom): 4 day PCR gelFigure 10 (Top Right): 1 week PCR gel

Acknowledgements❖ Dr. Cynthia Wagner for facilitating the class❖ BIOL 340L Grad TA’s Jesse Fox and Jong Park for general lab

help❖ BIOL 340L Classmates

α-JLA 2043kDa

Figure 5 (Top Left): 18 hour western blot gelFigure 6 (Bottom): 4 day western blot gel Figure 7 (Top Right): 1 week western blot gel

KEYLane 1: 100bp DNA LadderLane 2: 0% Octanol Heads (OH)Lane 3: 0% Octanol Tails (OT)Lanes 4-9: 25H, 25T, 50H, 50T, 75H, 75T A: Actb (Beta-actin) C: HSP60 (control)

Actb

Actb

Actb

Lanes: 1 2 3 4 5 6 7 8 9

Heads Tails

HSP601,622 bp

1,162 bp