c elegans

Cell Signaling – Levamisole Sensitivity Notes

Before Lab

  • READ the entire lab below

  • Write a clear, concise PURPOSE STATEMENT in your own words in your lab notebook.

  • Write a HYPOTHESIS:
      - How will loss of specific gene products affect the sensitivity to an anti-parasitic drug?

During Lab

  • Document step by step what you did during the lab.

  • Make note of any changes to the protocol or any mistakes that you made.

Introduction

  • You are engaged in a lab that studies cell signaling in the nematode Caenorhabditis elegans.

  • During your rotation project, you determined that the unc-11 and unc-63 gene products are involved in cell signaling pathways that facilitate locomotion in C. elegans.

  • Next Goal: Investigate whether the unc-11 and unc-63 gene products are components of the levamisole-sensitive nicotinic acetylcholine receptor.

Background

C. elegans Cell Signaling Involved in Locomotion

  • C. elegans locomotion is facilitated by four longitudinal bands of muscle, arranged sub-dorsally and sub-ventrally.

  • Mechanism of Movement:
      - Muscles flex and relax alternatively, generating dorsal-ventral waves along the body, propelling the organism.
      - For sinusoidal movement, contractions of dorsal and ventral muscles are out of phase (Driscoll and Kaplan, 1997).
      - Example: To turn dorsally, dorsal muscles contract while opposing ventral muscles relax.

  • Locomotion’s sinusoidal pattern is regulated through interactions between excitatory and inhibitory motor neurons.

  • Motor Neuron Processes:
      - Comprise presynaptic regions that form neuromuscular junctions and provide input to postsynaptic regions.
      - Synaptic Transmission Process:
        - Involves neurotransmitter synthesis and packaging into synaptic vesicles.
        - Calcium-regulated fusion of vesicles at nerve terminals.
        - Release of neurotransmitters.
        - Activation of postsynaptic receptors via neurotransmitter binding.
        - Recycling of vesicle membranes/proteins via clathrin-mediated endocytosis (Harris, 2001; Nonet, 1999).

  • This signaling cascade requires a variety of proteins for effective operation and successful locomotion in the organism.

Acetylcholine (ACh) as a Neurotransmitter

  • Primary Neurotransmitter: ACh acts as the major excitatory neurotransmitter within C. elegans and is essential for viability.

  • Mechanism of Action:
      - Secreted by presynaptic cholinergic neurons.
      - Activates nicotinic ACh receptors (nAChRs) on postsynaptic cells.

  • Nature of nAChRs:
      - nAChRs are heteromeric (composed of different subunits encoded by a significant gene family) and are classified as ionotropic receptors.
      - These receptors mediate the fast-synaptic action of ACh present on nerve and muscle cells.
      - Limited functional studies have been conducted on C. elegans nicotinic receptors, with extensive research focused on subunits of the nAChR (the levamisole receptor) at the neuromuscular junction.

  • Levamisole:
      - An anthelminthic drug that acts as an nAChR agonist, leading to open channel blockade.
      - Induces behavioral effects such as spastic paralysis of body wall muscles.
      - Researches have used levamisole-resistant mutants to identify genes encoding nAChR subunits and those impacting receptor function (Brown et al, 2006; Rand and Nonet, 1997).

  • Significance of Levamisole Receptor:
      - The levamisole receptor and a levamisole-insensitive nAChR provide balanced contributions to excitatory neurotransmission at the neuromuscular junction.
      - Null mutants of the levamisole receptor exhibit partial locomotion impairments.

C. elegans as a Model for Parasitic Nematode Infections

  • Despite not being a parasitic organism, C. elegans serves as a model to understand the effects of levamisole and other anthelmintic drugs.

  • Context of Parasitic Infections:
      - Parasitic worm infections pose significant threats to humans, animals, and crops, impacting global health and economic stability.

  • Anthelmintic Drug Classes:
      - Developed to treat parasitic infections, with three major classes:
        - Benzimidazoles
        - Macrocyclic lactones
        - Nicotinic agonists (Sleigh, 2010)

  • Laboratory Focus:
      - Effects of nicotinic agonist levamisole on C. elegans locomotion, specifically in unc-11 and unc-63 mutations.
      - Your research will assess if the absence of unc-11 or unc-63 gene products changes levamisole sensitivity.

Experimental Protocol

Examination of Levamisole Sensitivity

  • Goal: Are UNC-11 and/or UNC-63 gene products components of the levamisole-sensitive nAChR?

Experimental Conditions to Observe

  1. Preparation: Obtain and label three multiwell plates for each worm genotype:
       - Each plate contains one experimental well and one control well.
       - Experimental Well: + levamisole
       - Control Well: NO levamisole

  2. Add M9 Buffer:
       - Add 3 ml of M9 buffer to both wells per plate.

  3. Worm Transfer:
       - Using a worm pick, transfer 10 worms from the appropriate condition into each of the respective wells (total of 10 worms per well).

  4. Movement Check:
       - After transferring worms, check all for movement and record the total number in each well.

  5. Levamisole Addition:
       - Add 100 µl of Levamisole stock solution (50 mM) to the experimental well, mix gently, & start the timer.

  6. Paralysis Scoring:
       - Wait 30 seconds and record the number of paralyzed worms in each well.
       - Note: Paralyzed animals should exhibit no movement and appear hypercontracted (shorter).
       - Make observations swiftly as the timer continues.

  7. Scoring Interval:
       - Score paralysis every 30 seconds for a total of 5 minutes.

  8. Data Recording:
       - Enter observations and data in your lab notebook (see sample data table).

  9. Repeat:
       - Steps 4-9 for each experimental condition.

Sample Data Table for Levamisole Sensitivity

  • Table Format for Data Recording:
      - Worm Types: Wild Type (N2), UNC-11 mutant, UNC-63 mutant
        - Columns to Include:
          - Time (seconds)
          - Number paralyzed
          - Total number
          - Percentage paralyzed

WT Control

Levamisole

Time (sec)

# paralyzed

Total #

% paralyzed

0

30

60

90

120

150

UNC 11-mutant Control

Levamisole

Time (sec)

# paralyzed

Total #

% paralyzed

UNC 63-mutant Control

Levamisole

Time (sec)

# paralyzed

Total #

% paralyzed

Data Analysis

Levamisole Assay

  1. Graphing:
       - Plot % paralysis vs time for each experimental group (treated and untreated with levamisole) on separate lines.
       - Each line represents a different experimental condition.

  2. Analysis Questions:
       - a. Differences between WT worms in levamisole vs. control buffer? What significance does this hold?
       - b. Differences between mutant worms treated with levamisole vs. control buffer? What significance?
       - c. Differences in paralysis between WT and mutant in control treatment? What does this imply?
       - d. Differences between WT and mutant in levamisole treatment? What significance?

References

  • Alberts, B, et al. (2002) Cell Communication (Accessed August 10, 2010), in Molecular Biology of the Cell, 4th edition. NY: Garland Science.

  • Brown, LA, et al. (2006) International Journal for Parasitology 36: 617.

  • Campbell, NA, et al. (2008) Biology, 8th Edition. San Francisco: Pearson Benjamin Cummings.

  • Driscoll, M, Kaplan, J. (1997) Chapter 23 Mechanotransduction (Accessed August 17, 2010) in Riddle, DL, et al. eds. C. elegans II, 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press.

  • Dykxhoorn, DM, et al. (2003) Nature Reviews 4:457.

  • Harris, TW, et al. (2001) Traffic 2:597.

  • Lowry, R. (1999) Concepts and Applications of Inferential Statistics (online textbook) http://faculty.vassar.edu/lowry/webtext.html (Accessed August 17, 2010).

  • Moerman, DG and RJ Barstead. (2008) Briefings in Functional Genomics and Proteomics 7: 195.

  • Nonet, ML, et al. (1999) Molecular Biology of the Cell 10:2343.

  • Rand JB and Nonet ML. (1997) Chapter 22 Synaptic Transmission (Accessed August 17, 2010) in Riddle, DL, et al. eds. C. elegans II, 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press.

  • Riddle DL, et al. (1997) Chapter 1 Introduction to C. elegans (Accessed August 17, 2010) in Riddle, DL, et al. eds. C. elegans II, 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press.

  • WormClassroom. http://www.wormclassroom.org. (Accessed August 10, 2010)