Organization of the Cell : Organization of the Cell
Lecture Topics : Lecture Topics Cells: the basic unit of life
Methods for studying cells
Cell organization
Organelles
Cytoskeleton
Extracellular matrix
The Cell Theory : The Cell Theory Cells are the smallest parts of any organisms that can still be clearly identified as either being that organism, or originating in that organism - Matthias Schleiden (botanist) and Theodor Schwann (zoologist)
All cells are derived from cells - Rudolf Virchow
All cells can be traced back to a common ancient ancestor - August Weissman (an extension of Virchow’s concept)
Pasteur Refuted Spontaneous Generation : Pasteur Refuted Spontaneous Generation Popular concept: Life was thought in some cases to arise spontaneously.
e.g., Mice from piles of hay; maggots from rotting meat
Pasteur tested this:
Knew that broth would grow fungus, bacteria
Knew that he could kill microorganisms by boiling
Tested to see if the growth of microbes was due to the broth spontaneously growing them, or if they came from the environment
Pasteur’s Experiment : Pasteur’s Experiment Used ‘retorts’: flasks with long arms he could seal off
Boiled (sterile) broth in sealed retort
If seal maintained, or if the retort outlet (arm) very long, no growth
If the retort arm was broken, opening retort to the environment, then growth
Therefore, microorganisms grew from previous organisms in environment
Cell Function and Structure Is Conserved Across All Living Systems : Cell Function and Structure Is Conserved Across All Living Systems All cells compartmentalize their contents away from the environment with a plasma membrane
All cells contain a DNA-based information system
All cells maintain an internal homeostasis
All cells have metabolism: use energy from environment to build structure
All cells reproduce: grow and increase in numbers
All cells respond to stimuli
All cells move
All cells evolve and adapt to environment
Cells Are Small : Cells Are Small Most cells ~1mm (1/106 m) to 1 mm in diameter
Some are bigger
The Small Size of Cells Is Beneficial : The Small Size of Cells Is Beneficial The smaller the cell, the greater the surface:volume ratio
Improved transmembrane transport for given cell volume
Cell Structure Reflects Function : Cell Structure Reflects Function Cellular structures have evolved because of a need for a particular function, for example:
Neurons have axons to transfer information to other cells
Sperm have whiplike tails to enable them to swim
Eggs have large quantities of nutrients to aid early embryonic development
Early History of Microscopy : Early History of Microscopy Hooke first observed cells in cork (book Micrographica, 1665) and named them after small study rooms used by monks. He did not realize the tiny ‘rooms’ were living structures
His microscope very rudimentary - poor quality image
Antony van Leewenhoek: Dutch eyeglass maker, remarkable skill at grinding lenses
Made unusual small microscopes – very good quality
(1670) First reported observing living unicellular organisms he called “animalcules”
His skill not passed on
Microscopes greatly improved in late 19th century and early 20th century
Improvements in Microscopes : Improvements in Microscopes Improved glass making and grinding allowed investigators to clearly identify living cellular structures
Combination of improved chemical knowledge with microscopy: revealed cellular details
Improved fixation techniques (killing and immobilizing), staining, and embedding techniques gave rise to histology and cytology (study of cellular structure)
Magnification and Resolution : Magnification and Resolution Magnification is important
Ratio of observed size to actual size
Allows observer to see detail
Maximum magnification about 1000X
Resolution is very important
Resolution is smallest distance two objects can be separated and observed to be distinct
Critical to producing usable, interpretable image
Resolution is dependent on wavelength of light
Shorter wavelength, better resolution
Thus, blue light provides better resolution than red light
Best possible resolution with modern microscope is 0.2 mm (2 X 10-7 m) using green (560 nm) light
Stereo (Dissecting) Microscopes : Stereo (Dissecting) Microscopes
Allow user to see 3D structure – useful for surgery, dissection
Very well-made, adjustable magnifying system
Good for studying overall structure
OK for studying only the largest cellular structures (not organelles)
Transmitted Light Microscopes : Transmitted Light Microscopes Brightfield (oldest modern design; typical student microscope)
Depends on staining for best contrast
Phase contrast
Enhances contrast by enhancing destructive and constructive interference in the image
Designed in 1940s by Otto Zernicke
Allows study of living cells without stain
Differential interference
Enhances contrast by enhancing interference, but has fewer artifacts in image than phase contrast
Sees gradients in thickness and refractive characteristics
Has very clear 3D image, thin focal plane, very good detail
Designed by Nomarski (different versions by other scientists) in early-mid 1960s
Allows study of living cells without stain
Fluorescence Microscopes : Fluorescence Microscopes Fluorescence microscope
Much like transmitted light microscope, but sends light down objective to excite fluorescent molecules
Excitation light causes fluorescent molecules to glow
Reveals location of specific molecules: investigator usually labels the molecules
Cells may be fixed (dead) or alive; depends on application of technique
Confocal fluorescence microscope
Computer-driven, special fluorescent light microscope – some use laser to illuminate specimen
Provides exceptionally clear fluorescent images
Electron Microscopes : Electron Microscopes Use beams of electrons boiled off hot filament to form images
Have tall column down which electron beam moves – air pumped out to allow electrons to move unimpeded
Electron beam accelerated down column by high voltage between filament and cap with a hole … beam continues down evacuated column by inertia
Very high magnification: up to 500,000X
For most biological applications, up to ~100,000X
Very good resolution: ~ 1 nm (10-9 m)
Comparison of Light and Electron Microscopes : Comparison of Light and Electron Microscopes
Transmission Electron Microscopy (TEM) : Transmission Electron Microscopy (TEM)
Sample is thinly sectioned (sliced), plastic-embedded cells/tissues stained with metal atoms
Electron beam absorbed, deflected by metal in sample
Image is a shadow: electron beam “shadow” cast on phosphorescent ceramic plate or film view with ‘binoculars’
Film developed as normal black and white negative, and printed to produce the positive print
Scanning Electron Microscopy (SEM) : Scanning Electron Microscopy (SEM) Looks at surface of specimen
Surface coated with metal
Primary beam hits and scans over surface
Detector ‘reads’ secondary electrons, scans surface
Voltage signal is read as function of time and played out on special high-resolution ‘slow scan’ TV set (= cathode ray tube)
Biochemical Tools to Study Cells : Biochemical Tools to Study Cells In order to study cellular chemistry, must gain access to cellular compartments:
Break cells open
Disrupt in a grinder/glass homogenizer
Resulting mixture of organelles and cytoplasm is the homogenate
Centrifugation uses high gravitational force – G-force – to separate different cellular compartments
Centrifugation : Centrifugation Used to separate cellular components
Homogenate is poured into strong centrifuge tube which is revolved rapidly
Heavier organelles form a pellet; fluid above the pellet is the supernatant
Supernatant is centrifuged at greater G-force, a new supernatant is recovered and subjected to greater G-force, and so forth
Separates out cellular components based on size and density
Centrifugation : Materials to be separated (such as a homogenate of cell components) are loaded into centrifuge tubes
Tubes are loaded into rotor (‘hinged bucket’) and spun by powerful motor.
Heavier material is driven by centrifugal force to the outer periphery of the circle described by rotating centrifuge tubes. Centrifugation
Differential Centrifugation : Differential Centrifugation Homogenization disrupts cells, releases organelles
Centrifugal force separates larger, heavier components from smaller, lighter components
Stepwise process using increasing centrifugal force in each successive step
Density Gradient Centrifugation : Density Gradient Centrifugation Pellet placed on top of a gradient of solute, usually sucrose
Lowest density of sucrose at top of tube – highest at bottom
Steps or continuous gradient of higher sucrose down the tube
Fractions of the homogenate migrate to form band at the position which matches density
Other Methods : Other Methods Column chromatography: methods for separation and purification of molecules, usually using beads
By size – passing through a molecular mesh
By charge – ionic molecules bind to charged surfaces of beads
Electrophoresis: movement of molecules in electrical field
Determine size of molecules
Can examine ‘native’ (unperturbed) molecules
Can ‘break’ molecules and examine denatured molecules
Proteins, nucleic acids, and carbohydrates can be studied
Techniques to identify molecules
‘Western’ blotting – proteins identified with antibodies
Nucleic acids identified with ‘Southern’ and ‘Northern’ blotting
Techniques to assay function
Identify unique activity – e.g. - breakdown of hydrogen peroxide by catalase (catalase was the first fully characterized enzyme)
Cells and Non-Cells : Cells and Non-Cells Life has made enormous changes to the Earth
Earth is covered with many different forms of Life and non-living materials that are of biological origin, but are not alive
It is very important to understand the difference between actively living, and biological, but non-living, things
Living things are made of cells, ALWAYS!
Viruses: NOT alive, NOT cells : Viruses: NOT alive, NOT cells Viruses are not alive
They are dependent on cells for their existence
They are a compartment (protein capsid) that contains a bit of nucleic acid as an information system
Cells are Compartmentalized : Cells are Compartmentalized All cells are bounded by a plasma membrane
Protects cellular reactions from the environment
All cells have cytoplasm
Fluid compartment of the ‘body’ of the cell called cytosol
Internal organelles found within the cytosol
Cells have internal compartments
Keep different reactions apart
Keep compatible reactions together
Eukaryotic cells have membrane-bounded organelles
Membranes are very important to the cell
Cellular membranes
Transport materials in/out of cell
Locate/hold reactions (in/on organelles)
Distinguishing Features of the Two Major Cell Types : Distinguishing Features of the Two Major Cell Types
Bacteria (Prokaryotes) : Bacteria (Prokaryotes) Cell wall
Plasma membrane
Cytoplasm
Nucleoid
DNA, genome
Mesosome
Internal membrane
Energy metabolism
Ribosomes
70S
Flagellum
Single protein, flagellin
An Animal Cell : An Animal Cell A pancreatic cell that makes digestive enzymes
A Plant Cell : A Plant Cell A photosynthetic plant cell
Eukaryotic Endomembrane System : Eukaryotic Endomembrane System Nucleus: Birthplace of the endomembranes
DNA held in chromosomes: cellular library of information
Nucleolus: birthplace of ribosomes
Endoplasmic reticulum (rER and sER): cell’s factories
Rough endoplasmic reticulum (rER) makes secreted proteins and integral membrane proteins (IMPs) and modifies proteins
Smooth ER (sER) makes lipid molecules, such as steroids, and adds lipids to proteins
Golgi apparatus
Modifies proteins made in the rER
Lysosomes
The “destroyer”: proteolytic, lipolytic enzymes, acid pH
Vacuoles and microbodies
Storage & concentration of cellular waste, nutrients, and enzymes
The nucleus : The nucleus Nucleus
Chromatin
Condensed heterochromatin
Noncondensed
Nucleolus
Birthplace of ribosomes Double-layered membrane of nuclear envelope
Nuclear pores
Endomembranes and Secretion : Endomembranes and Secretion The nucleus produces the Rough Endoplasmic Reticulum (RER), a system of flattened membranes
The RER makes proteins that are secreted or inserted into membranes
The RER passes its protein products via transport vesicles to the Golgi Apparatus, which are also flattened membrane systems, which processes them . . .
And sends them to various locations, including the plasma membrane
Note that proteins produced inside the RER stay inside the Golgi and transport vesicles, but are expressed on the surface of the plasma membrane
Ribosomes : Ribosomes Made of 3 RNA strands and 75 different proteins
Free: suspended in cytosol
Bound: associated with rough endoplasmic reticulum
Assemble proteins by dehydration synthesis
Smooth Endoplasmic Reticulum : Smooth Endoplasmic Reticulum The smooth endoplasmic reticulum (SER) is tubular, unlike the RER.
It receives membrane from the RER.
Is tubular, (unlike the RER, which consists of flattened membranes)
Mitochondria are shown here also
Non-Endomembrane Organelles : Non-Endomembrane Organelles Mitochondrion
Energy organelle
Aerobic respiration
Derived from an ancient bacterium engulfed by a heterotrophic prokaryotic ancestor of the eukaryotes (’urkaryote’)
Ancient bacterium became an endosymbiotic organism
Chloroplast
Light-energy collection organelle
Makes oxygen
Photosynthetic bacteria and chloroplasts changed the world
Production of oxygen changed ancient atmosphere
Derived from bacterium, like mitochondrion
Mitochondria : Mitochondria Sausage-shaped
Outer and inner membranes
Inner membrane infolded to form cristae
Chloroplast : Chloroplast Similar in structure to mitochondrion
Photosynthetic membranes in thylakoids
Mitochondrial and Chloroplast Functions Are Complementary : Mitochondrial and Chloroplast Functions Are Complementary
The Cytoskeleton : The Cytoskeleton A system of filaments within the cytoplasm; the “internal materials” of the cell
Provide structure for the cell
Bind the cell together
Provide “highways” within the cell
Tracks for movement of organelles
Microfilaments (7 nm dia)
Actin 43 kDa
2 strands, twisted like string
Microtubules (little tubes) (25 nm dia)
Tubulin 50 kDa
13 protofilaments, arranged in a cylinder
Intermediate filaments (10-12 nm dia)
Many different types 40-~100 kDa
e.g., keratin (hair, fingernails, skin surface)
Cytoskeleton: Actin Filaments : Cytoskeleton: Actin Filaments F-actin (filamentous actin microfilament)
Two strings of G-actin (globular actin) attached end-to-end
7 nm wide
Made of actin: 43,000 molecular weight
Filament hydrolyses ATP as it grows
F-actin very common - forms shape of cells; allows amoebae to move, to take up food; facilitates muscle contraction, cell division, and cell anchorage
Muscles are Actin- and Myosin-Based Machines : Muscles are Actin- and Myosin-Based Machines Muscle sarcomere
Myosin is an actin ‘motor’
Myosin (200,000 MW) makes thick filaments in muscle.
Myosin hydrolyzes ATP, pulls on the F-actin filaments to contract muscle
Myosin and actin slide past each other as contraction occurs
Microtubules : Microtubules Microtubule (right)
Made of tubulin: 50,000 molecular weight
Alpha and beta isoforms of tubulin join together to make dimers, then dimers join to make the tubule
GTP hydrolyzed to GDP and phosphate as the tubule grows
In picture to right, tubulin dimers move through; dimers add at (+) end, fall off at (-) end: called treadmilling
Microtubules Are Important for Eukaryotic Cell Division : Microtubules Are Important for Eukaryotic Cell Division
Microtubule Motors : Microtubule Motors Dyneins: 1, 2, or 3 “heads” that cleave ATP
Run along microtubules
Large, complex: up to 25 polypeptides, 2,000,000 MW
Run toward base of cilia (“minus end-directed”)
Make cilia wiggle; can move very fast
Pull vesicles with nutrients
Kinesins: 1 or 2 heads that cleave ATP
Also run along microtubules
Small, only 2 types of polypeptides, total of 4: 150 kDa
Tend to be slow
Drag vesicles over microtubules
Move in opposite direction to dyneins (“plus end directed”)
Anatomy of Eukaryotic Cilia and Flagella : Anatomy of Eukaryotic Cilia and Flagella 0.25 m diameter
Made of 9 outer doublet microtubules: special double microtubules
2 central MTs
Very complex,
~300 proteins!
Dynein arms link doublets together, push tipward and drive the bending of the cilium
Kinesin : Kinesin Walks on the microtubule; carries a vesicle
Hydrolyses ATP to cause movement
Vesicle is adapted to the kinesin molecule by intermediate complex
Centrioles : Centrioles Also based on MTs
Within the MT- organizing center (MTOC)
9 triplet MTs + 0 MTs in center
Near nucleus
Important in mitosis
Intermediate Filaments : Intermediate Filaments Long fibrous proteins associate side-by-side, then end-to-end
No overall polarity, unlike actin and microtubules, which are polarized with specific longitudinal organization
Very strong and stable
Intermediate Filaments : Intermediate Filaments
Extracellular Matrix : Extracellular Matrix A system of proteins and carbohydrates outside of the cell.
Includes cell walls.