Cellular Biology: From Microtubules to Motor Proteins
Kinesin Movement
Kinesin walks with a limp, as determined by the “hand-over-hand” model instead of the inchworm model. The limp theory suggests that the neck of the kinesin acts like a spring, and successive steps produce coiling and uncoiling of this spring. Kinesin is powered by the hydrolysis of ATP -> ADP + P.
Microtubules
Microtubules are a component of the cytoskeleton found throughout the cytoplasm. They are composed of small alpha and beta tubulins, which form rows of tubulins, creating a larger diameter structure (13 protofilaments surrounding the center). Microtubules have an inner diameter of 12nm and an outer diameter of 24nm. They are often referred to as “cellular train tracks.”
Natural Selection
Natural selection is a process where any population with excessive offspring and variability in traits will see some traits improve chances of survival, therefore being passed along.
Bacteria are Prokaryotic Cells
Bacteria are prokaryotic cells that contain:
- Plasma membrane (phospholipid bilayer, same functions as cell membrane (barrier for cell transport))
- Ribosomes (make protein, float within cytosol)
- Cell wall (lends structural properties)
- Flagellum (helically shaped, rotates for motion like a propeller)
Ebola
Ebola has a genome of 19,000 nucleotides long.
Plant Cells
Plant cells differ from animal cells in several ways:
- Chloroplasts: responsible for photosynthesis
- Cell walls: provide structural support
- Vacuoles: filled with inorganic molecules and water to support the cell
Other important organelles in plant cells include:
- Rough ER: site of ribosomes (thus protein production)
- Smooth ER: makes cellular products like hormones and lipids
- Golgi Complex: processes and bundles proteins and lipids
- Centriole: helps with cell division
- Microfilaments: provide support to structure, flexible and strong (actin filaments)
Cytoskeleton
The cytoskeleton is a complex network of structures within the cytoplasm that gives the cell shape and mechanical resistance.
Ramachandran Plot
A Ramachandran plot shows limitations in bond angles of polypeptide chains. Beta sheets are located in the top left, alpha sheets in the middle left/center. Weak non-covalent bonds contribute to protein folding.
Lysozome
Lysozome catalyzes hydrolysis and is abundant in secretions. Its reaction can be represented as: S+E -> ES -> EP -> E + P
Methods
The maximum magnification for light microscopy is 1000x. Specimens for TEM are often ultrathin sections (100nm thick). SEM has achieved a resolution of up to 50pm. The advantage of Ultra High Voltage TEM is that the resolving power of the instrument increases with voltage. Thick specimens are easily penetrated, leading to decreased chromatic aberration and decreased damage from specimen heating. The objective lens collects a cone of light rays. The condenser lens focuses a cone of light rays onto each point of the specimen.
Chromatography
There are different types of chromatography:
- Separation: A solution is placed into the top of a liquid and travels at different speeds throughout the liquid, creating clear layers.
- Ion-Exchange: Positively charged beads are placed in a solution. Negatively charged ions attach to the beads, while positively charged ions flow freely.
- Gel-Filtration: Porous beads are used to collect smaller-sized particles through channels, while larger particles do not fit and flow freely.
SDS-PAGE
In SDS-PAGE, a sample is loaded on top of a vertically placed gel between two charged buffer solutions. It is used for the separation of proteins based on molecular weight. SDS brings proteins from a folded structure to a linear structure, which masks their charge. SDS-coated proteins have the same charge-to-mass ratio, so separation will be solely dependent on molecular weight.
Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) is a way to amplify DNA strands. It includes repeated cycles of heating and cooling. Primers, which contain sequences complimentary to the target region, form. PCR employs a heat-stable enzyme like TAQ polymerase, which is able to assemble a new DNA strand using free nucleotides. Amplifications can be equated to genetic cloning.
Actin Filaments
Actin filaments are the smallest subunit involved in the cytoskeleton, 4-7nm thick. Nucleation begins with a few G-actin molecules forming a nucleus. The process follows these steps: Nucleation (lag phase) -> elongation (growth) -> Steady State. It looks like a first-order response during the growth phase before reaching a steady state. The polymerization process begins when salt is added to G-actin molecules.
Dynamic Instability of Microtubules
Dynamic instability of microtubules refers to the coexistence of assembly/disassembly at the end of microtubules. At the plus end, lateral forces binding tubulin together are much weaker. The length of a microtubule is incredibly unstable, ranging from 2-50 microns over the course of an hour.
Fast Axonal Transport
Fast axonal transport is responsible for the movement of mitochondria, lipids, etc. over the cell’s volume. Axons, known as nerve fibers, transmit information to different neurons, muscles, and glands.
Molecular Organization of Kinesin
Kinesin looks like two little balls at the end (globular heads), a coiled alpha helix, and a light chain at the end.
- Kinesin moves from the negative to the positive end of the microtubule.
- ATP hydrolysis is used for the movement of kinesin.
- The average speed of kinesin movement is 500nm/s.
- Kinesin can produce about a 5pN force.
- Speed changes as a function of ATP available.
Biomolecular Motors – Myosin
Myosin is another ATP-dependent motor protein, best known for its role in muscle contraction.
- The head region binds to the actin filament and “walks along the filament.”
- The neck domain acts as a linker and lever arm to transduce force.
- The tail domain may play a role in regulating motor activity.
Fasciculus
A fasciculus is a bundle of skeletal muscle fibers surrounded by connective tissue.
Pentose in RNA
The pentose sugar in RNA is ribose.
Order of Processes in Terms of Energy Content
From smallest to largest energy content:
- Thermal motion at room temperature
- ATP hydrolysis under cellular conditions
- Breaking of a C-C bond
- Complete oxidation of a molecule of glucose
Beta Sheet
A beta sheet is a common structural motif of proteins with different sections of the protein’s polypeptide chain running alongside each other. They are held together by hydrogen bonding of atoms in the polypeptide.
Non-Covalent Bonds in Proteins
Two non-covalent bonds that are critical for proteins are electrostatic bonding and van der Waal’s forces. Hydrogen bonding also plays a significant role.
Origins of Replication in Human Cells
The minimum number of origins of replication required to replicate the entire genome of a human cell is one per chromosome, for a total of 46.
Microtubules vs. DNA
Microtubules are stiffer than DNA.
Experiment to Measure Motor Force
A laser trapping assay can be used to record single motor force. To address the processive nature of the motor, immobilize it to a substrate and present a pre-tensed actin filament manipulated in a double laser trap. Carefully match the stiffness of the trap to the step size of the motor such that a single step reveals the full force of the motor.