Migration of Pigment Granules in Chromatophores from Cichlids (*Amatitlania nigrofasciata*).

# Migration of Pigment Granules in Chromatophores from Cichlids (*Amatitlania nigrofasciata*). * **IMPORTANT**: *You absolutely MUST wear closed-toe shoes and long pants or skirts to this lab. You will be handling toxins, and they are dissolved in an organic solvent that can diffuse through your skin. **You will not be allowed to participate in the lab without proper footwear.*** * **REMINDERS**: -You are expected to clean up before you leave the lab. (Your lab bench/station should be left in the same state you found it on arrival: garbage thrown away, microscope, turned off, reagents put away, etc.) *There is absolutely NO food or drink allowed in the labs at Colby College. Please leave any food or drink you bring with you in the hall outside of the lab.* ## Introduction: This week in lab we are putting aside the mammalian cells we have been working with up until now and switching systems. For the remainder of the semester you will be using chromatophores from Convict cichlids (*[Amatitlania nigrofasciata](https://www.fishbase.de/summary/Amatitlania-nigrofasciata.html)*), as a model system for studying cellular mechanisms controlling pigment distribution. Chromatophores, or pigmented cells, are found in the dermis of a variety of lower vertebrates and are responsible for the coloration of the organism. Each cell normally contains many granules, each surrounded by a membrane. These granules can be clustered toward the middle of the cells or dispersed throughout the cells (See Figure 1). The former distribution leads to a pale coloration of the organism, while the latter leads to skin darkening. Changes from one pigment distribution to the other are under hormonal and neural control, so that the fish can change coloration in response to environmental factors. The directed movement of organelles also plays a prominent role in many important cellular processes such as axonal transport in neurons (transport of organelles to and from the cell body to the axon). Such movements are dependent on the cytoskeleton and much research has been devoted to elucidating roles of various cytoskeletal elements and the motors involved. Color changes are important survival mechanisms, as they allow fish to become less conspicuous to predators. Variation in color due to pigment granule migration can also be used in mating and territorial displays. This system is a nice illustration of how subcellular processes can affect entire organisms and populations. --- ![](https://i.imgur.com/6BS2NRO.png) ##### Figure 1: Schematic depiction of pigment granule aggregation and dispersal in a fish chromatophore. --- ## Objectives: One of the important goals of this laboratory is for you to connect changes that we can see macroscopically to subcellular events. Many of you may have seen fish or other organisms lighten or darken their coloration depending upon their environmental surroundings or mating and territory displays. * ### *How does this happen at the cellular level?* * ### *What are some of the mechanisms underlying these changes?* Another important goal of the laboratory is for you to gain first-hand experience devising and executing an experiment of your choosing. You will be asked to determine what experimental treatments you will do, what controls are important, how you will collect and analyze your data, etc. For this inquiry-based laboratory, there will be no detailed set of instructions that everyone will follow step by step. We will go over the basic procedures to be followed--for example, how to set scales up for observing the basic process of pigment aggregation and dispersion. Beyond this, you will plan your own set of observations and experimental variations, designed to probe the questions you are interested in addressing. Those questions could be focused on signaling – for example: *what are the signals?* *What are the second messengers?* *How do different signaling pathways interact?* Your questions could instead be focused on the role of the cytoskeleton: *do the granules move along microtubules?* *Microfilaments? (or both?)* *Can you inhibit the motors used to move the granules? Is movement dependent on ATP?* Alternatively your question might be motivated by your interests outside of this class for example: how does a specific chemical or environmental factor impact these processes. Maybe you have other questions. The rationale behind your experiment is entirely up to you. --- # Week 1: Introduction to the System The basic plan for today is to (1) familiarize ourselves with the scales/chromatophores/pigment granules from our Convict Cichlids and (2) to see if we can get pigment granules to aggregate and disperse. ## The Adrenergic System in Chromatophores Much of the research on fish pigmentation is based on studies using Tilapia mossambica as a model system (Rozdzial and Haimo 1986; Thaler and Haimo, 1990; 1992). These reports and others suggest that many fish dermal cells are responsive to the **alpha-adrenergic signal transduction pathway**. You likely have some firsthand experience with this pathway because it is the one that responds to epinephrine/adrenaline! Generally speaking, the adrenergic signaling pathway begins with the stimulation of an adrenergic receptor, of which there are at least 10 subtypes. These receptors differ in two ways: 1) generally speaking, the receptors have different ligand affinities and 2) each subtype is associated with different intracellular signal transduction machinery. As you can probably surmise from the complexity of this system, cells have the capacity to fine-tune their responses to stimuli. Additionally, cell signals are rarely all or nothing events. Cells receive signals, integrate them with other incoming signals and produce responses that are the sum of several stimuli. Keep this in mind as you work through this lab. There are several readings in the texts that will help you learn more about the signaling system we will cover them in detail later on in the course but if you would like to get a head start you might refer to the material covered in Chapter 16 of your textbook (Alberts, Bruce, et al. Essential Cell Biology. 6th ed.). --- ## Here is the basic protocol for the pigment granule aggregation/dispersal. --- ### You will be using two solutions: Frog Ringer’s Solution (FRG): 115mM NaCl, 2mM KCl, 1mM CaCl2, 0.1% glucose, 2mM Tris, pH 7 Perfusion Buffer (PB): 33mM potassium acetate, 30mM HEPES, 10mM EGTA, 5mM MgSO4, 0.5mM EDTA, 2.5% polyethylene glycol), pH 7.4 --- 1. Place a scale in a depression slide with FRG. 2. Observe the scale for 5 minutes. You can use a cell phone camera to take pictures at regular intervals (try 30-60 seconds and/or try a movie). 3. Using a piece of blotting paper (*eg.* a Kimwipe (c)), wick away the FRG and rinse with perfusion buffer (PB) by filling the depression slide with it, wicking it away and refilling the depression slide with perfusion buffer. Work very quickly so that the scale does not dry out. Observe the scale as above. 4. Repeat step 3, but use FRG instead of perfusion buffer; work quickly to exchange the perfusion buffer for FRG, and make your observations. --- ## *As you work through the lab today and each week. Make sure the purpose for your experiments and your conclusions are clear in your notebooks. In addition, please include:* 1. A flow chart or complete protocol of the steps you actually followed in this lab. 2. At least one table containing your data. Explain how you collected your data, and justify your reasons for doing it this way. 3. An explicit description of your controls – what they were controlling for and the results and how this affected your interpretation of your experimental samples. 4. Describe and document any statistical analysis you did on your data and explain your results. 5. Don’t forget to record observations during the experiments. Even if things are not working like you would have hoped, it is always important to record all the data. --- Since you will be conducting your own experiments and collecting your own data (which will differ from the rest of the class) it is **EXTREMELY** important that you have a clear understanding of what you are doing each week in lab and that you are keeping complete and thorough notes that you are able to refer back to from week to week. You will not be able to use data collected by another group and I do not have data for your experiment to give you. *Please plan accordingly!* **With this in mind, let's practice:** **In your notebook, begin by describing the practice experiment you are doing today:** * What is the hypothesis that you are testing in this experiment? * Which treatment condition(s) are your control? * Which treatment condition(s) are your experimental? * Make a FLOWCHART for your experiment. As part of this lab, you will need to decide how to collect and quantitate your data in a way that will allow you to make meaningful interpretations of what you see with your eyes. You will be able to collect images (still images or videos) of your cells. As you observe the chromatophores this week, take some time to consider how you might quantify the changes that you are seeing. **In your notebooks, DESCRIBE below how you might and quantify your data.** Your lab assignment (available on the course Moodle site and due before lab next week) will help you to work through one way you might do this using the ImageJ/FIJI software we have been working with this semester. (https://imagej.net/Fiji#Downloads). --- ### References *This laboratory was developed by Sue Ellen Gruber at Mount Holyoke College and Sally Nyquist and Kathryn Brady Toner based on experiments described primarily by Leah Haimo and colleagues at University of California, Riverside and Gary Borisy and colleagues at the University of Chicago. Further adjustments to the lab protocol were made by Elizabeth Vallen at Swarthmore College with helpful modifications that were suggested by Linda Silveira at University of Redlands and Lavinia Sheets and Bruce Schnapp at Oregon Health Sciences University as well as Christina Cota at Colby University.* 1. Nyquist, S. E., & Toner, K. B. (1997). Pigment Granule Transport in Chromatophores. 2. Rozdzial, M. M., & Haimo, L. T. (1986). Reactivated melanophore motility: differential regulation and nucleotide requirements of bidirectional pigment granule transport. The Journal of cell biology, 103(6 Pt 2), 2755–2764. 3. Thaler, C. D., & Haimo, L. T. (1990). Regulation of organelle transport in melanophores by calcineurin. The Journal of cell biology, 111(5 Pt 1), 1939–1948. 4. Thaler, C. D., & Haimo, L. T. (1992). Control of organelle transport in melanophores: regulation of Ca2+ and cAMP levels. Cell motility and the cytoskeleton, 22(3), 175–184.