September 25, 2000

Methods to Study Cells

• Cells, cellular organelles, their structure & function, and molecules within these organelles are studied using many different techniques. The limitations of each technique determine the reliability of the data.
• The more different techniques that are used to study the same phenomenon (getting similar data), the more reliable is that data.

Methods for Studying Cells

• Light Microscope
  • the wavelength of light restricts the resolution (to 200 nm)
  • objects need to be thin enough to pass light through them 
  • unless the objects are colored, they must be treated with specific colored dyes to reveal the parts of the cell of interest
• Transmission Electron Microscope (see page104 Figure 7.2a)
  •  the wavelength of an accelerated electron restricts the resolution (0.2 nm, theoretically, but 2 nm or greater in practice)
  • objects need to be extremely thinly sliced, made electron dense (usually by staining them with heavy metals), and then placed in a vacuum to observe them 
  • different very clever techniques have been developed to allow better observations of subcellular organelles and their components.
    • Freeze fracture ( Methods Box on page 138 in Chapter 8)
• Scanning Electron Microscope (see page104 Figure 7.2b)
  • resolving power 10 nm
  • the surface details are revealed after treating the surface with a heavy metal (e.g., gold)
• Cell Fractionation
  • Requires rupturing of cells (homogenization): What happens to membranes & cell contents when a cell is ruptured?
  • Separating the organelles to study their function apart from the rest of the cell
    • differential centrifugation
• Gel electrophoresis allows isolation of specific molecules and parts of molecules 
  • (DNA, RNA, proteins, sugars)
  • See Chapter 20 for details, Methods Box on page 374.
• Radioactive labeling of cells 
  • Methods Box, page 30;  Figure 20.4 page 369
  • One can measure different reactions in cells and the location and amounts of different molecules in cells by making different molecules  radioactive and then looking for the location of the radioactive label..
    • analyzing the specific molecules can be analyzed by liquid scintillation counting.

    • analyzing the specific location with cells by light microscope autoradiography OR electron microscope autoradiography

     

Structure & Function of Cellular Organelles

Key Ideas

• Prokaryotes: carry out many of the same functions as eukaryotes, but have no separate membrane-bound organelles.

• Eukaryotes: use membrane-bound organelles to create compartments. These compartments carry out separate cellular processes.

• Cells which are carrying out a specific function are enriched in the organelle(s) involved in that function.

Cellular Ultrastructure in Prokaryotes

• See Figure 7.4 and see Chapter 18 for more information.  Chapter 18 is not assigned at this time.

• These organisms have no nucleus, no mitochondria, no ER.

• These organisms have DNA in a nucleoid region, ribosomes, etc.

• Some prokaryotes have a mesosome (where the surface membrane is greatly infolded) providing a surface for the attachment of ribosomes, enzymes, and other molecules.

• Many prokaryotes have a cell wall (peptidoglycan = protein-containing sugar) and a polysaccharide capsule.

• The Central Dogma = DNA -> RNA -> protein  (and DNA replicates DNA)
• Cells which are carrying out a specific function, devote more of their cytoplasm and organelles to it.
  • For example: a cell whose function is secreting digestive enzymes (in the gut) will have a large amount of rough endoplasmic reticulum.
  • For example: a liver cell involved in removing anesthetic from the blood stream of a person recovering from surgery will have a large amount of smooth endoplasmic reticulum.
  • For example;  a cell with many motile cilia would have a large number of mitochondria (for energy production)
• Nucleus contains DNA & synthesizes rRNA, mRNA, & tRNA (TRANSCRIPTION)
  • DNA is contained within the nucleus as chromosomes (chromosomes = DNA + protein)
  • DNA contains the genes and serves as a template for tRNA and mRNA and some rRNA.
  • Is enclosed by a double membrane called the nuclear envelope.
  • Nuclear pores (~100 nm in diameter) control the entry and exit of ribosomal subunits, mRNA, tRNA, and the various proteins (including enzymes) that are used within the nucleus.
  • Nucleolus: 
    • is an area within the nucleus that contains DNA which codes for rRNA.
    • nucleolar DNA is the template for rRNA; ribosomes are synthesized here.
• Ribosomes are the site of Protein synthesis (TRANSLATION)
  • Ribosomal subunits are made in the nucleolus.
    • large subunit = 3 different rRNA molecules + 45 different proteins.
    • small subunit = 1 rRNA molecule + 33 different proteins.
    • What holds the proteins and rRNA together?
  • Small & large subunits are brought together by a mRNA molecule.
  • Protein synthesis starts immediately.
    • A SIGNAL part of the newly synthesized end of the protein determines whether the ribosome floats freely in the cytoplasm to continue synthesis of the protein --or-- whether the ribosome attaches to the rough ER.
    • Free polyribosomes = mRNA + several to many ribosomes
      • this is where cytoplasmic, soluble proteins are synthesized.
    • Rough ER = membranes of ER with ribosomes bound at specific sites.
      • this is where secretory proteins are synthesized
      • this is where  intrinsic (integral) membrane proteins are synthesized
      • this is where proteins that need to be moved into other organelles are synthesized
    • After the protein is synthesized the ribosomal subunits separate and are reused.
    • tRNA brings specific amino acids to the ribosome.
  • TRANSLATION (protein synthesis) occurs on ribosomes (RNA --> protein)
• Rough Endoplasmic Reticulum: synthesis of membrane and secretory proteins -TRANSLATION
  • Folding of proteins occurs in the Rough ER
  • Some sugars are added to specific amino acid R groups in the Rough ER.
• Smooth ER:
  • Further modifies proteins synthesized in Rough ER
  • Synthesis of lipids occurs in SER
  • metabolism of carbohydrates  (hydrolysis of glycogen)
  • Detoxification of drugs and anesthetics and poisons occurs here.
• Golgi apparatus:
  • Packages materials for secretion (release from the cell)
  • Molecules move from the cis to the trans face as they are modified
  • Modifies products of the Rough ER and Smooth ER
  • Adds molecular identification tags to proteins that tell the molecules where to go within the cell (e.g., the lysosome)
• Transport vesicles:  membrane sacs that move materials from one part of the cell to another.
• Lysosome:  membrane sac filled with digestive enzymes
  • In animals: contains enzymes that work best at pH=5; a proton pump moves H⁺ from the cytoplasm into the lysosome.
    • phosphatases: nucleic acids --> nucleosides + phosphate
    • phospholipases: phospholipids --> fatty acids + phosphate
    • glycosidases: polysaccharides --> monosaccharides
    • proteinases: proteins --> amino acids
    • Example: Tay Sachs disease affects brain function
      • a lipid-digesting enzyme is absent or inactive and this results in the accumulation of lipids within cells.
  • Plant vacuoles can act as lysosomes
    • can store proteins
    • can store pigments
    • may contain poisons (which kill or injure herbivores that eat them)
    • and some ions (K⁺, Cl⁺).
• Mitochondria: involved in cellular respiration = production of ATP in the presence of oxygen.
  • Have their own DNA, ribosomes, protein synthesis and enzyme (in the matrix compartment)
  • Matrix: contains DNA, ribosomes, protein synthesis and also the enzymes involved in the citric acid cycle.
  • Undergo mito-division (replication)
  • Cristae : contain enzymes and molecules involved in electron transport and oxidative phosphorylation.
  • Intermembrane space: usually has a high concentration of H⁺.
  • Outer membrane: has transport proteins to allow entry into the intermembrane space.
• Chloroplasts: Convert solar energy to chemical energy (sugars). The sugars are then used in glycolysis and cellular respiration to produce ATP.
  • Have their own DNA, ribosomes, protein synthesis and enzymes (in the stroma compartment)
  • Thylakoid membranes form sacs. The membranes contain chlorophyll, a hydrophobic molecule.
  • Stacks of thylakoid membranes form a granum.
  • A double membrane is present (inner membrane & outer membrane)
• The Cytoskeleton (pages 119-124) will be discussed when we talk about the cell cycle (Chapter 12)
• The Cell Surface
  • Plants, fungi, some protists, and some bacteria have ell walls
  • Many animal cells are surrounded by an extracellular matrix (see Figure 7.29)
    • components vary from cell to cell, but may contain different concentrations of:
      • collagen
      • proteoglycans
      • fibronectins (which bind to integrins - integral transmembrane  proteins)

• Which of the following cellular organelles would you expect to see using a light microscope?
  • nucleus,  Golgi apparatus, mitochondrion, chloroplast
• If you needed to determine whether a protein was present in the mitochondria of a cell or the nucleus of a cell, which techniques for studying cells would you use? Describe how you would go about determining the location of the protein and what results you would expect. Evaluate the reliability of that technique.
• What is the importance of mRNA in eukaryotic cells? Why is it necessary?
• Do plant cells have mitochondria? Why do they need/not need mitochondria?
• Which, if any, of the following is not a function of the endoplasmic reticulum:
  • synthesis of proteins exported from the cell.
  • synthesis of proteins used in the cytosol.
  •   modification of proteins
  •   lipid synthesis
• Where are ribosomes made? Of what are ribosomes constructed?
• Which of the following is the correct sequence of structures tracing the path of a protein from its site of synthesis to its point of export from the cell?
  • Smooth ER, Golgi apparatus, mitochondrion, vesicle
  • Smooth ER, vesicle
  • Rough ER, Golgi apparatus, vesicle
  • Rough ER, vesicle, Golgi apparatus, vesicle
• The mitochondrion of a cell:
  • makes ATP in the presence of oxygen.
  • synthesize proteins for export from the cell (secretory proteins)
  • has its own ribosomes (separate from those that are on the rough ER)
  • has a double membrane
• Cells of the pancreas, for example, that are making large amounts of secretory proteins would probably have many more free ribosomes in the cytoplasm than rough ER. True or False