c elegans 1

Cell Signaling - Locomotion Before Lab

  • Preparation Instructions:
      - Read the entire lab description thoroughly.
      - Write a clear, concise PURPOSE STATEMENT in your lab notebook.
      - Formulate a HYPOTHESIS regarding the following:
        - How the loss of specific gene products will affect locomotion in Caenorhabditis elegans.

During Lab

  • Documenting the Lab Procedures:
      - Record a step-by-step account of the lab activities conducted.
      - Note any alterations to the protocol or errors made throughout the lab.

Introduction

  • Lab Context:
      - You are a graduate student in a lab studying cell signaling between neurons in the nematode C. elegans.
      - The lab has generated worms with mutations in the unc-11 and unc-63 genes.
      - The objective is to determine if unc-11 and unc-63 gene products are necessary for the cell signaling pathways essential for locomotion in C. elegans.

Background

  • Importance of Cell Communication:
      - Cell communication is crucial for biological processes in both single-celled and multicellular organisms.
      - Cells communicate via chemical signals that can be:
        - Secreted or anchored molecules.
        - Require or do not require cell-cell contact.
        - Travel short or long distances.
        - Comprise a variety of organic or inorganic molecules.
      - General Process of Cell Signaling:
        1. Reception: The target cell receives the signal by the binding of a signaling molecule (ligand) to a specific receptor.
        2. Transduction: The bound receptor undergoes a conformational change, leading to an intracellular signaling cascade.
        3. Response: The intracellular signal results in various cellular responses, which may include changes in protein structure, function, or gene expression (Campbell, 2017).

  • Technical Understanding of Cell Signaling:
      - Signals induce a biological response and may lead to alterations in proteins or gene expression (Alberts, 2002; Campbell, 2008).

Study Methods in Cell Biology

  • Techniques to Study Cell Signaling:
      - Understanding cell signaling mechanisms is a key goal in cell biology.
      - Various techniques aid in examining the cell signaling pathways:
        - Assessing the impact of gene disruptions on signaling pathways.
        - Identifying functions of genes and associated proteins using mutation analysis.
        - Types of Genetic Screens:
          - Forward Genetic Screen: Identify organisms with mutant phenotypes to locate genetic loci responsible for those phenotypes.
          - Reverse Genetic Screen: Generate specific mutations in genes to observe resultant phenotypes (Moerman, 2008).
      - Mutation techniques include:
        - Homologous recombination.
        - UV light or chemical agents to induce mutations.
      - The analysis of resulting phenotypic changes can elucidate gene functions relevant to biological processes like cell signaling.

Caenorhabditis elegans as a Model Organism

  • Characteristics of C. elegans:
      - Belongs to the phylum Nematoda (roundworms and threadworms).
      - Smooth-skinned, unsegmented, cylindrical body;
        - Non-parasitic, free-living; survives in organic-rich soil by feeding on microbes (Riddle et al, 1997; WormClassroom).
      - Advantages as a Laboratory Model:
        - Non-parasitic nature allows safe laboratory use.
        - Small size (~1 mm) and simple diet facilitate mass production of phenotypically and genotypically specific worms.
        - Capable of producing 300-350 offspring per hermaphroditic self-fertilization and more through mating.
        - Short life cycle: 3 days from egg to reproducing adult; adults live 2-3 weeks.
        - Up to 10,000 worms can be cultivated on a single petri dish.
        - Well-characterized developmental lineage and genomic structure; constant cell lineage (959 cells in wild-type) and known cell positions.
        - Genome has 40% homologous sequences to the human genome, aiding the study of gene function and human diseases.

Cell Signaling in Locomotion

  • Muscular System of C. elegans:
      - Locomotion is achieved through four longitudinal bands of muscles located sub-dorsally and sub-ventrally.
      - Movement is generated via alternating flexion and relaxation of these muscles.
      - Dorsal-ventral waves propel the organism forward, requiring systematic contraction of dorsal muscles and relaxation of ventral ones to create a sinusoidal locomotion pattern (Driscoll and Kaplan, 1997).

  • Control Mechanisms:
      - Movement involves excitatory and inhibitory motor neurons, facilitating the alternating muscle contractions.
      - Major communication form between neurons and muscle cells is synaptic transmission, involving:
        - Synthesis of neurotransmitters and their encasing in synaptic vesicles.
        - Calcium-regulated fusion of vesicles at nerve terminals.
        - Release and binding of neurotransmitters to postsynaptic receptors.
        - Recycling of membrane and proteins via clathrin-mediated endocytosis (Harris, 2001; Nonet, 1999).
      - A multitude of proteins is required for proper signaling cascade and subsequent locomotion.

Experimental Protocol - Locomotion Assay

  • Objective: Determine involvement of UNC-11 and/or UNC-63 gene products in locomotion-related cell signaling pathways.

  • Experimental Conditions:
      | Experimental Condition | Type of Worm |
      |-----------------------|---------------|
      | Wild Type (N2) | N2 |
      | UNC-11 mutant | unc-11 |
      | UNC-63 mutant | unc-63 |

  • Protocol Steps:
      1. Label a multiwell plate for each condition.
      2. Add 3 mL of M9 buffer to a well.
      3. Utilize a worm pick to transfer one worm into the well; observe under a dissecting microscope, counting tail beats/min, documenting in the lab notebook.
      4. Repeat steps 2 and 3 for a total of 12 worms per condition.

  • Notes:
      - Handle worms gently with a worm pick.
      - A tail beat consists of a stroke and recovery (2 movements = 1 tail beat).
      - Avoid using dead worms in observations.
      - Limit buffer illumination to prevent overheating.
      - Shake the plate gently if worms cease movement.

  • Data Collection:
      - Table 1: Sample Muscular Performance (Thrashing Rate) Data Table (N2, Unc-11, Unc-63)

Data Analysis - Locomotion Assay

  • Bar Graph Representation:
      - Plot the mean thrashing rates of each group, including the Standard Error of the Mean (SEM).
      - SEM Explanation: Reflects the variability of the sample mean estimated from a population.

  • Calculating SEM and Standard Deviation:
      - Standard Deviation (STDV) Formula:
        STDV = rac{ ext{√} ext{Σ}(X - ar{X})^2}{n-1}
      - Standard Error of the Mean (SEM) Formula:
        SEM = rac{STDV}{ ext{√}n}
        - Where:
          - XX = individual thrashing rates
          - nn = sample size

  • Statistical Significance Testing:
      - Compare locomotion rates between:
        - Wild Type (N2) vs. UNC-11 mutant
        - Wild Type (N2) vs. UNC-63 mutant
      - Student T-Test Overview:
        - Essential for discerning whether observed differences are statistically significant.
        - Assumptions: Independent samples, equal sample sizes, equal variances.
        - The resulting T-test probability value (p-value) indicates whether groups represent the same population.
          - p-value Interpretation:
            - p<0.05p < 0.05: Differences considered statistically significant.         - p>0.05p > 0.05: Differences not statistically significant (Lowry, 1999).

Calculating T-Test in Excel

  • Procedure:
      1. Input data into two columns in Excel.
      2. Use the TTEST function with the syntax:
        - =TTEST(array1, array2, tails, type):
          - Array1: Data from column A.
          - Array2: Data from column B.
          - Tails: 2 (for two-tailed test).
          - Type: 2 (assuming equal variances).
      3. The function returns a value representing the probability that differences are due to chance.

  • Conclusion Interpretation:
      - If the returned probability value is less than 0.05, the differences between groups are statistically significant.
      - If the returned probability value is greater than 0.05, the differences are not statistically significant.

References

  • Alberts, B., et al. (2002). Cell Communication in Molecular Biology of the Cell, 4th edition, NY: Garland Science.
  • Brown, L. A., et al. (2006). International Journal for Parasitology, 36: 617.
  • Campbell, N. A., et al. (2008). Biology, 8th Edition, San Francisco: Pearson Benjamin Cummings.
  • Driscoll, M., Kaplan, J. (1997). Mechanotransduction in C. elegans II, 2nd edition, Cold Spring Harbor Laboratory Press.
  • Dykxhoorn, D. M., et al. (2003). Nature Review, 4:457.
  • Harris, T. W., et al. (2001). Traffic, 2:597.
  • Lowry, R. (1999). Concepts and Applications of Inferential Statistics.
  • Moerman, D. G., Barstead, R. J. (2008). Briefings in Functional Genomics and Proteomics, 7:195.
  • Nonet, M. L., et al. (1999). Molecular Biology of the Cell, 10:2343.
  • Rand, J. B., Nonet, M. L. (1997). Synaptic Transmission in C. elegans II, 2nd edition.
  • Riddle, D. L., et al. (1997). Introduction to C. elegans II, 2nd edition.
  • WormClassroom. (Accessed August 10, 2010).