Course : PGPathshala-Biophysics Paper 3 : Cellular and Molecular Biophysics Module 17 : Methods to study Cell motility Content Writer: Dr. Ruby Dhar, DBT-BioCARE , AIIMS NEW DELHI METHODS TO STUDY CELL MOTILITY: Movement is a major characteristic of living organisms, and can take the form either of movements of cells or of movements within cells themselves. In biology, motility is the ability to move spontaneously and actively, consuming energy in the process. Most animals are motile but the term applies to unicellular and simple multicellular organisms, as well as to some mechanisms of fluid flow in multicellular organs. Motility is genetically determined, but may be affected by environmental factors. There are several methods to understand the motility of cells under the influence of various conditions and this chapter gives a overview of various technologies, developed so far to understand the process of cell motility. INTRODUCTION: Most cells in the body are stationary, but many of these exhibit dramatic changes in their morphology — the contraction of muscle cells, the elongation of nerve axons, the formation of cell-surface protrusions, the constriction of a dividing cell during mitosis. Cell motility is needed for some vital physiological procedures amid improvement, for example, cell movement amid gastrulation, tissue recovery and embryological advancement. A type of cell motility includes the dynamic transport of membranous organelles inside of the cytoplasm. This type of development is needed for fitting association of the cytoplasmic substance, and the redistribution of metabolites, hormones, and different materials inside of the cell. Cell motility is one of the crowning achievements of evolution. Primitive cells were probably immobile, carried by currents in the primordial milieu. With the evolution of multicellular organisms, primitive organs were formed by migrations of single cells and groups of cells from distant parts of the embryo. Perhaps the most subtle movements are those within cells — the active separation of chromosomes, the streaming of cytosol, the transport of membrane vesicles. These internal movements are essential elements in the growth and differentiation of cells, carefully controlled by the cell to take place at specified times and in particular locations. Advancement in technology development had enabled us to monitor the motility of single cell to group of cells both in 2D and 3D method, including live cell tracking. OBJECTIVES: 1. Motility as a vital part of living organisms 1.1 Different modes of motility 1.2 Flagellar movement 1.3 Amoeboid movement 1.4 Gliding movement 1.5 Swarming movement 2. Difference between motility and Brownian motion 3. Methods to study bacterial motility 3.1 Hanging drop method 3.2 Band formation method 3.3 Bailey’s method 4. Studying sperm motility 5. 2-D Single Cell locomotion Assays 5.1 Colloidal Gold migration assay 5.2 Live cell Tracking 5.3 Dunn Chamber 6. 2-D methods to study group cell motility 6.1 Chemotaxis under agarose method 6.2 Microcarrier Bead assay 6.3 Ring assay 6.4 Aggregate migration assay 6.5 Wound healing assay 7. 3-D Cell Motility assays 7.1 Boyden Chamber/Matrigel invasion assay 7.2 Collagen invasion assay 7.3 Invasion in an organ culture 8. Microscopy in studying cell motility 9. Microfluidics to understand cell motility 1. MOTILITY AS A VITAL PART OF LIVING ORGANISMS Our liaison with cell migration, as humans, begins shortly after conception, accompanies us throughout life, and often contributes to our death. Although migratory phenomena are apparent as early as implantation, cell migration orchestrates morphogenesis throughout embryonic development. During gastrulation, for example, large groups of cells migrate collectively as sheets to form the resulting three- layer embryo. Subsequently, cells migrate from various epithelial layers to target locations, where they then differentiate to form the specialized cells that make up different tissues and organs. Analogous migrations occur in the adult. In the renewal of skin and intestine, fresh epithelial cells migrate up from the basal layer and the crypts, respectively. Migration is also a prominent component of tissue repair and immune surveillance, in which leukocytes from the circulation migrate into the surrounding tissue to destroy invading microorganisms and infected cells and to clear debris. The importance of cell migration however, goes far beyond humans and extends to plants and even to single-celled organisms. All cells can be considered motile for having the ability to divide into two new daughter cells. 1.1 At the cellular level, different modes of motility exist: flagellar motility, a swimming-like motion (observed for example in spermatozoa, propelled by the regular beat of their flagellum, or E. coli, which swims by rotating a helical prokaryotic flagellum) amoeboid movement, a crawling-like movement, which also makes swimming possible gliding motility swarming motility 1.2 Flagellar movement: A flagellum (/fləˈdʒɛləm/; plural: flagella) is a lash (whip)-like appendage that protrudes from the cell body of certain prokaryotic and eukaryotic cells. The primary role of the flagellum is locomotion, but it also often has function as a sensory organelle, being sensitive to chemicals and temperatures outside the cell. Flagella are organelles defined by function rather than structure. Large differences occur between different types of flagella; the prokaryotic and eukaryotic flagella differ greatly in protein composition, structure, and mechanism of propulsion. However, both can be used for swimming. An example of a flagellated bacterium is the ulcer-causing Helicobacter pylori, which uses multiple flagella to propel itself through the mucus lining to reach the stomach epithelium. An example of a eukaryotic flagellate cell is the mammalian sperm cell, which uses its flagellum to propel itself through the female reproductive tract 1.3 Amoeboid movement: Amoeboid movement is the most common mode of locomotion in eukaryotic cells. It is a crawling-like type of movement accomplished by protrusion of cytoplasm of the cell involving the formation of pseudopodia and posterior uropods. The cytoplasm slides and forms a pseudopodium in front to move the cell forward. This type of movement has been linked to changes in action potential; the exact mechanism is still unknown. Examples: amoeboids, slime molds and some protozoans such as Naegleria gruberi, as well as some cells in humans such as leukocytes. Sarcomas, or cancers arising from connective tissue cells, are particularly adept at amoeboid movement, thus leading to their high rate of metastasis 1.4 GLIDING MOTILITY: Gliding motility is a type of flagella-independent translocation that allows the microorganism to, glide smoothly along the surface; without the aid of propulsive organelles on the outer membrane . Gliding motility and twitching motility both allow microorganisms to travel along the surface of low aqueous films, but while gliding motility is smooth, twitching motility is jerky and uses the pili as its mechanism of transport. EXAMPLES: Apicomplexa, a Eukaryota parasite, travelling at fast rates between 1-10μm a second when the Myxococcus xanthus glide at a rate of 5μm a minute 1.5 SWARMING MOTILITY: Swarming motility is a rapid (2–10 μm/s) and coordinated translocation of a bacterial population across solid or semi-solid surfaces and is an example of bacterial multicellularity and swarm behaviour. This multicellular behavior has been mostly observed in controlled laboratory conditions and relies on two critical elements: 1) the nutrient composition and 2) viscosity of culture medium (i.e. % agar). One particular feature of this type of motility is the formation of dendritic fractal-like patterns formed by migrating swarms moving away from an initial location. Although the majority of species can produce tendrils when swarming, some species like Proteus mirabilis do form concentric circles motif instead of dendritic patterns. 2. DIFFERENCE BETWEEN MOTILITY AND BROWNIAN MOTION: Motility is different from Brownian movement and both have the following characteristics: MOTILITY: Unique and directional movement of individual cells from one point to another BROWNIAN MOTION: Aimless and unidirectional movement Oscillating and quivering motion 3. METHODS TO STUDY BACTERIAL MOTILITY (FLAGELLUM DEPENDENT) : There are three maim methods to study bacterial motility, as stated below: 1. Hanging drop method 2. Observation of motility band 3. Bailey method, staining of the flagellum 3.1Hanging drop method: This method is used to stain live, unstained bacterial cells, suspended in water or broth. Take a clean glass slide and apply paraffin ring, adhesive tape ring to make circular concavity. (This step is not needed if a glass slide with depression is available). Hold a clean coverslip by its edges and carefully dab Vaseline on its corners using a toothpick. Place a loopful of the broth culture to be tested in the center of the prepared coverslip. Turn the prepared glass slide or concavity slide upside down (concavity down) over the drop on the coverslip so that the vaseline seals the coverslip to the slide around the concavity. Turn the slide over so the coverslip is on top and the drop can be observed hanging from the coverslip over the concavity. Place the preparation in the microscope slide holder and align it using the naked eye so an edge of the drop is under the low power objectives. Turn the objective to its