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Questions and Answers
What is the temperature required for cultivation in a CO2 incubator?
37 ºC
What is the pH level used with phenol red as an indicator dye?
7.4
What do primary neuron cultures from brain cortex require for growth? (Select all that apply)
Primary cell cultures derived from normal animal cells have a limited life span.
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Microdissection techniques allow selected cells to be isolated from __________ slices.
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Match the cell surface modification with its description:
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What is the process called where cells are broken up and their components and organelles are separated for observation?
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Which mechanical methods can be used for cell disruption during homogenization? (Select all that apply)
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In cell fractionation, a finer degree of separation can be achieved by layering the homogenate on top of a dilute salt solution in the centrifuge tube.
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What is the complex internal structure characteristic of cilia called?
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______ is the first step in most cell fractionation methods to separate components that differ greatly in size.
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What is the arrangement of microtubules in a cilium or flagella known as?
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What was the first cell-free system to carry out protein synthesis in vitro?
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Ciliary movement is based on the contraction of microtubules. True or False?
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Which organelles can be separated by ultracentrifugation? (Select all that apply)
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In Kartagener's syndrome, patients have normal number of spermatozoa but no _____
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What is the main function of tight junctions?
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Which type of junction provides the strongest points of cell adhesion that provide mechanical binding?
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Desmosomes are abundant in tissues that are not exposed to mechanical stress.
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Hemidesmosomes resemble __________ morphologically.
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What is the main structural protein of ECM?
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What is the function of Hyaluronic acid (HA) in tissues?
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Elastin and fibrillin provide tissue ________.
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Match the following ECM proteins with their functions:
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What is the role of integrins in hemidesmosomes?
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What is the structure that anchors loops of intermediate filaments in hemidesmosomes?
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Gap junctions allow the rapid passage of molecules 1.2 nm in diameter between cells.
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Mutations in connexin genes cause ________.
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Match the following adhesion proteins with their respective functions:
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What is the function of collagen in the extracellular matrix?
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What are the two main types of protein translocation according to the content?
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The signal peptide is about _$ amino acids long and appears at the beginning of the polypeptide chain.
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Properly folded proteins are retained in the Golgi apparatus.
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What enzyme catalyzes the rearrangement of disulfide bonds in the GER lumen?
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Match the following modifications with their descriptions:
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What disease is caused by mutations in the gene for ADAMTS13?
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What is the primary role of VWF (von Willebrand Factor) in hemostasis?
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Stem cell fate is influenced by the stiffness and topography of the ECM.
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Smooth Endoplasmic Reticulum (SER) is primarily involved in the biosynthesis and transport of _____?
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Match the following with their functions: 1) GER (Granular ER) 2) SER (Smooth ER)
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What are the short amino acid sequences called in ER resident proteins?
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What causes ER dysfunction and proteotoxicity in the ER?
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The ER stress response helps cells survive under ER stress conditions.
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Which pathway eliminates misfolded proteins in the ER by the ubiquitin-proteasome system? The ER associated __________ pathway.
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Match the function of unfolded protein response (UPR) with the correct description:
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Study Notes
Cell Fractionation and Analysis
- Cell fractionation is a method of breaking up cells into their components, such as organelles and macromolecules, to study their processes.
- Cells are disrupted using various methods, including osmotic shock, ultrasonic vibration, and grinding, which break many of the cell membranes.
- Organelles, such as nuclei, mitochondria, and lysosomes, are left intact and can be separated from the rest of the cell components.
- Homogenization is the process of breaking open cells, and it involves the use of chemical, enzymatic, or physical methods.
- Centrifugation is used to separate the cell components based on their size, density, and surface properties.
Steps of Subcellular Fractionation
- Homogenization: breaking open cells to release their components.
- Differential centrifugation: separating the components based on their size and density.
- Density gradient centrifugation: further separating the components based on their density and surface properties.
- Collection of fractions: collecting the separated components for further analysis.
- Analysis of fractions: studying the separated components to understand their functions and properties.
Cell-Free Systems
- Cell-free systems are fractionated cell extracts that maintain biological functions.
- These systems are used to study molecular mechanisms involved in cellular processes.
- Examples of cell-free systems include:
- Isolated myofibrils from skeletal muscle cells that contract upon the addition of ATP.
- Cell-free systems that carry out protein synthesis, DNA replication, and transport along microtubules.
Cell Theory
- Cells are the fundamental units of both structure and function in all living organisms.
- All living organisms are composed of cells and their products.
- Cells arise only from preexisting cells.
Methods for Examining Cells
- Examination of living cells using a microscope.
- Examination of killed and preserved cells using a microscope.
- Cell culture: growing cells in a controlled environment to study their behavior and properties.
- Cell fractionation: breaking up cells into their components to study their processes.
Cell Culture
- Cell culture is a method of growing cells in a controlled environment to study their behavior and properties.
- Cells are isolated from tissues and grown in a culture medium that supplies the necessary nutrients.
- Cell culture is used to study cell growth, differentiation, and responses to various stimuli.
Applications of Cell Culture
- Study of cancer cells and their behavior.
- Study of cell-cell interactions and communication.
- Study of cell nutrition and the effects of environmental factors on cell behavior.
- Production of hybrid cells by fusing cells from different species.
- Production of monoclonal antibodies using hybridoma cells.
- Genetic analysis and gene mapping using hybrid cells.
Mechanical Micromanipulation
- Mechanical micromanipulation is a method of manipulating cells using microsurgical procedures.
- This method is used to study cell elasticity, viscosity, and the dependence of cell function on the nucleus.
- Microinjection of substances into cells can be used to study cell behavior and function.
- This method is also used in in vitro fertilization and the production of transgenic animals.### Epithelial Cells and Polar Differentiation
- Epithelial cells show polar differentiation, with apical, lateral, and basal surfaces
- Apical surface is also known as luminal or free surface
- Epithelial cells exhibit modifications on their surfaces, including microvilli, cilia, and flagella
Microvilli
- Finger-like projections from the cell surface
- Function: increase the surface area for absorption
- Localizations: intestinal epithelium, proximal tubule of the kidney, and epithelium of the gall bladder
- Structure: enclosed in an extension of the plasma membrane, containing a bundle of straight parallel filaments (actin filaments)
Cilia and Flagella
- Hair-like protrusions from the cell surface
- Function: to move fluid over the surface of the cell
- Localizations: surface of epithelial cells of the upper respiratory tract, uterine tubes, and efferent ducts
- Structure: complex internal structure, including a 9+2 microtubule arrangement (axoneme)
- Cilia beat in a rhythmical wave-like manner, sliding microtubule mechanism
Mechanism of Ciliary Movement
- Sliding microtubule mechanism: ciliary movement is based on the sliding of doublet microtubules relative to one another
- Dynein arms are essential for motility of cilia and flagella
- Kartagener's syndrome: a genetic disease characterized by the absence of dynein arms, leading to immotile sperm and respiratory tract disorders
Basal Body
- A dense granule at the base of each cilium
- Cylindrical structure, 0.2 μm wide and 0.4 μm long
- Composed of nine sets of triplet microtubules
- Function: organizes the axoneme microtubules, plays a role in the development of left-right asymmetry
Flagella
- Longer than cilia, propels sperm
- Same internal structure as cilia (axoneme 9+2)
- Different type of movement (undulating wave type)
- Less in number, with a protective function
Cell-Cell Junctions
- Found on lateral surface of epithelial cells
- Functions: cell-to-cell attachment, formation of barriers, and cell-to-cell communication
- Classified into three functional groups: occluding junctions, anchoring junctions, and channel-forming junctions
Tight Junctions (Occluding Junctions)
- Located just below the apical surface
- Function: seals neighboring cells together, prevents passage of molecules, and separates apical and basolateral domains of the plasma membrane
- Structure: consists of an anastomosing network of protein strands, composed of transmembrane proteins (claudin, occludin)
- Important for the blood-brain barrier and blood-testes barrier### Blood-Brain Barrier
- Separates blood from nervous tissue, preventing substances from passing through
- Formed by endothelial cells of capillaries in the brain and spinal cord, which are joined by continuous tight junctions
- Inflammatory states like meningitis can reduce the integrity of the blood-brain barrier
Anchoring Junctions
- Also known as adhering junctions
- Composed of two classes of proteins: transmembrane adhesion proteins (cadherins) and intracellular anchor proteins (vinculin, alpha-actinin, catenin, desmoplakin)
- Functions:
- Cell-cell adhesion
- Binding of cytoskeleton to the cell surface
Adherens Junctions
- Also known as zonula adherens
- Surrounds the entire periphery of epithelial cells near their apical surface
- Forms adhesion belts that link neighboring cells together
- Maintains the physical integrity of the epithelium
- Found in heart muscle, connecting cardiomyocytes to one another
- Actin filament bundles are attached to cadherins through intracellular anchor proteins
Desmosomes
- Button-like points of tight adhesion
- Anchorage sites for intermediate filaments
- Strongest points of cell adhesion, providing mechanical binding
- Most abundant in tissues exposed to mechanical stress (epidermis, heart muscle)
- Contain plaque-shaped structures on the cytoplasmic face of the junction
- Cytoplasmic plaque proteins (plakoglobins, desmoplakins, plakophilins) link cadherins to intermediate filaments
Hemidesmosomes
- Resemble desmosomes morphologically
- Cell uses hemidesmosomes to attach to the basal lamina
- Cell-matrix adhesion proteins, integrins, bind to laminin protein in the basal lamina
- Permeability of hemidesmosomes is regulated by posttranslational modification of integrins and environmental changes
Channel-Forming Junctions
- Gap junctions
- Allow for the free interchange of ions and larger molecules between cells
- Physiologically important for the coordination of function among groups of cells
- Composed of assemblies of six connexins
- Permeability of gap junctions is regulated by posttranslational modification of connexins and environmental changes
Mutations in Connexin Genes
- Cause human disease, such as recessive inherited deafness
- Affect the transport of K+ in the epithelia supporting the sensory hair cells in the ear
Anchoring Junctions
- Can be subclassified according to the cytoskeletal element involved
- Actin filament attachment sites: adherens junctions and focal adhesions
- Intermediate filament attachment sites: desmosomes and hemidesmosomes
Adhesion Proteins
- Cadherins (cell-cell adhesion, Ca2+ dependent)
- Integrins (cell-matrix adhesion)
- Immunoglobulin-superfamily members (cell-cell adhesion, Ca2+ independent)
- Selectins (mediate transient cell-cell adhesions in the bloodstream)
Selectins
- Mediate transient cell-cell adhesions in the bloodstream
- Bind to carbohydrate molecules on the surface of leukocytes and platelets
- Play a major role in initiating the adhesion of leukocytes and platelets during the inflammatory and hemostatic responses
Extracellular Matrix
- Comprises proteins and polysaccharides secreted by cells
- Fills the spaces between cells and binds cells and tissues together
- Provides structural integrity, mechanical strength, and resilience
- Consists of different types of matrices, depending on the tissue
Matrix Structural Proteins
- Tough, fibrous proteins (collagen, elastin)
- Provide structural integrity, mechanical strength, and resilience
- Embedded in a gel-like polysaccharide ground substance
Collagen
- Most abundant protein in the extracellular matrix
- Secreted mainly by fibroblasts
- Accounts for up to 30% of the total proteins in the human body
- Comprises a family of 28 genetically distinct proteins
- Characterized by the presence of a repeated sequence of three amino acids (Gly-X-Y)
- Forms a triple helix structure
Mutations in Collagen Genes
- Cause diseases such as chondroplasia, osteogenesis imperfecta, Alport syndrome, Ehlers-Danlos syndrome, and dystrophic EB
- Contribute to osteoarthritis and osteoporosis
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Description
Learn about the methods of cell fractionation and analysis of molecules in medical biology, including the isolation of organelles and macromolecules.
