Midterm Flashcards
Central dogma of life
DNA->RNA->Protein
Cells are the simplest structure that can support the
process of the genetic information flows from DNA to
RNA (transcription) and from RNA to protein
(translation)
Viruses rely on the cell to reproduce themselves and
thus they are not considered living organisms
Model organisms of cell biology
• E.coli perfect for studies of DNA replication, transcription, translation
• Yeast Saccharomyces cerevisiae – simple model of the eukaryotic
cell
• Drosophila melanogaster developmental biology and genetics
• Snails, zebrafish – neuroscience
• Mice, rats, pigs etc…
Cells biology scale
<1 micron to ~100 microns
Light Microscopy
Advantages: simple and the
least invasive.
Disadvantage: can mostly be used for morphological studies, can tell what kind of processes are happening inside the cell
Transmission Electron microscopy
Advantage: highest resolution possible, broad range;
Disadvantage: only fixed cells, takes a long time to prepare sample, possible artifacts of preparation
Fluorescence microscopy
The most versatile and widely used method. Can be applied to both live and fixed cells. Allows to investigate structure as well as biological activity of the cells.
Cell electrophysiology
Direct measurement of the electrical activity of the cell.
Particularly useful for studies of excitable cells: Neurons, Cardiac, Muscle cells.
Can measure: membrane potential; integral cell ion current; single-channel ion current.
Respirometry
Real time measurement of cell energy
metabolism by measuring consumption of the
oxygen and production of the carbon dioxide.
Allows to measure: rates of ATP production;
energetics capacity of the cell; rates of glucose and
fatty acids metabolism; overall mitochondrial health
and fitness.
Role of Cell Membrane
Barrier between the cell and the environment or cell organelles
- Maintain Ionic and molecular homeostasis
- Signal transduction
- Energy production
- Cell growth and motility
- Molecular synthesis
Different membranes of the cell
• Plasma (cell) membrane: signal transduction; cell
movement; import/export of small molecules
• Nucleus: storage of DNA material
• Mitochondria: energy production; cell death; calcium
signalling
• Endoplasmic reticulum: protein synthesis; calcium
signalling
• Golgi: protein and membrane assembly and trafficking
• Peroxisome, lysosome, vesicles:
compartmentalization
Fluid Mosaic Model
Fluid lipid bilayer forms a matrix dependent on the amphipathic nature of lipids
• Lipids can diffuse laterally and rotate
• Proteins are either peripheral or integral and may diffuse laterally and rotate
accounted for:
• Varied protein (20-75%) & lipid content
• Asymmetry of both protein & lipid (head size)
• Rapid diffusion of proteins in membrane (modulated by cholesterol which increases packing to decrease fluidity)
• High electrical resistance
• Impermeability of polar substances
Membrane Lipids
Amphipathic: has both hydrophobic &
hydrophilic regions, as in phospholipids or detergents
• Spontaneously form micelles or bilayers & reseal
• 109 molecules/small cell
– Phospholipids: 14-24 C tail; asymmetrical; unsaturated
(C=C) increase fluidity by decreasing packing
– Cholesterol: modifies fluidity by increasing packing
– Glycolipids: only on external face
• Asymmetrical with little flip-flop across bilayer
• Primary diffusion barrier
• High electrical resistance: 106 Ω/cm2
MOST ENERGETICALLY STABLE IN SPHERICAL FORM - NOT PLANAR
Phosphatidylcholine
most common phospholipid in cell membranes
Lipid Classification
Glycerolipids – based on glycerol:
- Phosphatidylcholine (PC), - Phosphatidylethanolamine (PE) - Phosphatidylserine (PS) - Phosphatidylinositol (PI)
Sphingolipids – based of sphingosine (can be phospholipids, but not only, can be glycolipids)
-Sphingomyelin
Glycosphingolipids (contain sugar)
Sterols
-Cholesterol
Unique for mitochondria – cardiolipin (4 tails)
Lipid Bilayer Movements
- Lateral diffusion
- Flexion
- Rotation
- Flip-flop (rare)
Increases to Membrane Fluidity
1) INCREASE in unsaturation of lipids
2) DECREASE in lipid chain length
3) DECREASE in Cholesterol
4) INCREASE in Protein Content
Asymmetric lipid distribution in the membranes
Phospholipids and glycolipids are distributed asymmetrically in the plasma membrane
bilayer
Hexagonal head groups are sugars on glycolipids, which are restricted to the outer leaflet.
Cholesterol is distributed almost equally in both monolayers.
Scramblase – random transfer of lipids
Flippase – specific transfer of lipids
Membrane Lipid Rafts
Induced by Cholesterol
a lateral segregation in a lipid mixture
• a more ordered structure
• helps to segregate proteins lateral distribution
Membrane proteins perform several different functions
Transporters
Anchors
Enzymes
Receptors
Cell Cortex
Formed by Spectrin & actin
Provide mechanical strength for plasma membrane
Allows cells to change actively shape and to move
Restrict the diffusion of proteins within the membrane
Glycocalyx
•Many extracellular membrane proteins & lipids have sugars (Glycocalyx) that protect and lubricate surfaces & involved in cell–cell recognition.
Membrane Permeability
Only permeable to SMALL, NONPOLAR molecules (such as oxygen, CO2 etc.) but most other molecules
require participation of the specialized transport systems
General classification of the transport mechanisms
Simple diffusion – does not require any protein – just going through the lipid bilayer
Channel – the protein which forms the pore, that allows free (but selective!) flow of the molecule(s)
Transporter – protein which takes the molecule on the one side and releases on the other (never opens as a “pore”)
ACTIVE transporter – can carry molecules against the concentration and/or electrical gradient.
Transport Mechanisms
Uniport – unidirectional transport of specific molecule (ion)
Symport – co-transport of two different molecules
Antiport – two molecules are transported in the opposite directions
Active Transporters (also called “Pumps”)
Coupled pump uses the energy gradient of the “coupled” molecule
-
ATP driven – uses energy released during the hydrolysis of ATP into ADP and phosphate*
- ATP driven pump is responsible for establishing ion gradients in excitable cells such as Na+ and K+ or Ca+
Light driven – direct use of light energy
Ion Channels
passive selective transport of ions driven
by electrochemical gradient
Ion channels are integral proteins in the lipid bilayer, can be open or closed.
Open channel is characterized by specific selectivity.
Channels can be classified by selectivity (what ion they allow to go through) and gating (what makes them
transfer from open to closed state and back).
EXAMPLE IS NEURAL COMMUNICATION
Ion channels – general classification
• By gating
Ligand gated
Voltage gated
Mechanosensitive
• By selectivity
Sodium
Potassium
Calcium
Chloride
Cationic
Anionic
Large pores (technically not “channels”)
Nernst equation
the force tending to drive an ion across a membrane is made up of two components:
one due to the electrical membrane potential and one due to the electrical membrane potential and one due to the concentration gradient of the ion.
At equilibrium, the two forces are balanced and satisfy a simple math relationship give by the Nerst Equation:
V = 62 log10 (Co/Ci)
V= membrane potential in milivolts Co = concentration outside Ci = concentration inside
Action Potential
Na conductance shows a fast,
transient increase in response
to depolarization
K conductance shows a slower,
more sustained increase
Functional studies of ion channels
can be performed using patch-clamp approach
Channel structure Investigation
using X-ray crystallography or cryoelectron microscopy
Targeted Protein synthesis
Ribosomes are targeted to the ER only
after protein signal (targeting) sequence
becomes synthesized.
SO…
There is a common pool of ribosomal subunits sitting in the cytosol, If there is no targeting sequence synthesized, protein synthesis will occur via the free ribosomal cycle. HOWEVER, if there is a targetting signal, the ribosomal subunits will bind to the rough ER for protein synthesis.
Coordinated with protein translocation - mainly on the
Ribosomes bound to rough ER
Soluble protein synthesis on ER
Following synthesis, the soluble proteins are released into the lumen of the ER
Signal peptide (signal sequence) is cleaved by signal peptidase and remains in the ER membrane
Membrane protein synthesis on ER
Following synthesis proteins remain in the ER membrane***
Stop transfer sequence (hydrophobic sequence) keeps protein imbedded in membrane, seperating lumen and cytoplasmic sides of the protein
Posttranslational protein modification in ER
Posttranslational modification – chemical
modification of the protein that is not coded
by DNA-RNA. (COVALENT BONDING OF DIFFERENT MOLECULES AFTER PROTEIN IS ALREADY MADE)
Many dozens exist and can
involve many amino acids and many types of
“attachments”
ex. Glycosylation – attachment of a sugar
residue like with the Glycocalyx
Golgi Apparatus
the main destination for the proteins
made in ER
Protein synthesized in ER, then they need to be processed in the golgi apparatus and get
there via vesicles. Golgi vesicles then take finished proteins for either exocytosis or endocytosis
Golgi apparatus structure
Golgi consists of many flatten membrane enclosed sacs: “cisternas” (3 to 20 cisternas in every stack of Golgi)
Has two sides: cis (entry side) and trans (exit side)
Cis – close to the ER; Trans – close to the plasma membrane
Golgi apparatus vesicular transport
Proteins entering ER (cis) are either moved
trough the stack or returned back to the ER (if
they have ER retention signal)
Proteins exiting from Golgi (trans) either go to
the lysosomes or cell surface
Golgi is the place where further sugar protein
modification occurs
SO…it can either undergo UNREGULATED exocytosis in which newly snythesized soluble proteins are transported via a transport vesicle made up of newly synthesized plasma mem. lipids/proteinsto the cell membrane for constitutive secretion
OR…
it can go through REGULATED exocytosis where the secretory where the proteins are transported into a SECRETORY vesicle - which stores the proteins until an extracellular signal (hormone or neurotransmitter) signals for the secretion of these proteins. thus, the signal transduction allows for the regulated secretion of the proteins out of the cell.
Lysosomes
Primary cites of intracellular digestion -
Includes acid hydrolases nucleases proteases glycosidases lipases phosphatases sulfatases phosolipases
The acidic environment of the lysosome is maintained by ATP dependent proton pump
There’s transporters on the membrane of lysosome that also transport metabolites out of the lysosome after the enzymes have broken them down.
o Nearly all of these enzymes in the lysosome are protected from being broken down themselves because they got lots of glycosyl modification; the
(mannose 6-phosphate) that protectthem from being
broken down by other lysosomal enzymes.
Lysosomal digestion pathways
Phagocytosis – digestion of
bacteria
Endocytosis – digestion of the
extracellular compounds
Autophagy – digestion of the
intracellular compounds
ER and Calcium Signaling
- ER plays a critical role in calcium signalling during muscle contraction
- ER in muscle is called SR (Sarcoplasmic Reticulum)
- SR is highly specialised to perform calcium pumping and release cycles
MUSCLE CONTRACTION CAN NOT PROCEED WITHOUT PARTICIPATION OF ER!!!!!!!!
Also, essential role of ER in neuronal calcium signalling
(TG-thapsigargin - drug that blocks calcium uptake in the ER)
Mitochondrial protein synthesis and import
• ~2000 proteins make mitochondrial
proteome
• Only 20-30 are coded by the mitochondrial
DNA
• Most of the mitochondrial proteins are
made in cytoplasm and imported into the
mitochondria
Mitochondria have their
own complete machinery for protein synthesis but only for vey few proteins
Mitochondrial DNA is maternally inherited:
it allows us to track different
inheritance because there is almost no DNA material mixing. You can track by mother inheritance and parental information in terms of origin of
different genomic lines.
Mitochondrial protein import machinery
The whole protein is made in the
cytoplasm and then it gets imported into mitochondria in an unfolded form through the protein translocator in the outer membrane and protein translocator in inner membrane.
• Proteins that are destined to the mitochondria generally have this signal sequence which can be recognized by this transporting protein. Then
can be translocated and unfolded and get into the matrix where they complete protein folding and the signal peptide gets cleaved and recycled.
• But what is not really well understood if you look at mitochondrial protein only a few proteins have this signal protein. By looking at the
protein we cannot determine if its mitochondrial or not. If we have a signal sequence we can but if it doesn’t, you cannot be for sure whether its
mitochondria targeted or not.
- proteins targeted to the (other than matrix) mitochondrial compartments have different targeting sequences
- many mitochondrial proteins do not have any obviously recognizable mito-targeting sequence.
Mitochondria (definition)
structures within cells that convert the energy from food into a form that cells can use” (NIH-2009)
- Mitochondria produce energy in form of ATP
- Mitochondria and calcium signaling
- Mitochondria – storage of pro-apoptotic molecules (they are key master regulator of cell death which is apoptosis.)
-Mitochondria – major source of ROS (Reaction Oxygen Species)
»_space;> Oxygen species are major signaling molecules that guide a lot of intracellular processes. Mitochondria are the place where most of these species are synthesized which can signal throughout the cell. Mitochondria are critical for that. Nobody knows how they do this.)
Diseases/Disorders/Conditions involving Mitochondria
• Diabetes Type II • Cardiomyopathy • Aging • Cancer • Parkinson’s Disease • Alzheimer's • Huntington’s
Cellular Energy Metabolism
Mitochondria are central in producing energy in eukaryotic cells especially in cells that rely on oxidative phosphorylation for energy.
• Two major pathways: glucose utilization and pathway for fatty acid utilization. Those are two types of food our cells can receive and process. A
majority of foods when it comes to the tissues come in the form of glucose or fat associated with proteins.
• One pathway goes from glucose, which is sugar, through pyruvate and then into the mitochondria. Then is the critic acid cycle (TCA) producing NADH and then end up in ETC which utilizes oxygen and eventually leads to the production of ATP
• The other pathway also eventually leads either to TCA, producing NADH or directly producing NADH and then to electron transport chain and again the
synthesis of ATP.
KEY METABOLITES
• GLUCOSE and FATTY ACIDS are the starting points.
- Then PYRUVATE is the central end product of glycolysis.
- ACETYL CO A is the major intermediate entering the TCA cycle from either side.
- NADH is major donor of protons and electrons to the electron transport chain and the end product is ATP.
Energy Production by Mitochondria
Outer membrane is permeably to the ions and small molecules
**98% of the body’s oxygen is used***
1) NADH is produced by TCA cycle
2) NADH donates electron and proton to the respiratory chain
3) Electrons travel through the respiratory chain towards complex IV and this is
coordinated with proton exiting from the matrix
4) Membrane potential is generated as a result of proton efflux
5) ATP is produced in ATP synthase when protons flow back into the matrix
Coupling of the Oxidation and Phosphorylation (Oxidative Phosphorylation)
- Oxidation – movement of the electrons from NADH to the oxygen which results in generation of the membrane potential
- Oxidation can be coupled to the Phosphorylation: Membrane potential is used for attachment of the phosphate to ADP and production of ATP
- Membrane potential can be used for:
1) ion transport
2) heat generation
3) pathological and physiological “leak” of the membrane
Mitochondrial Ion Transport
Roles of the mitochondrial ion channels:
• calcium uniporter – regulation of energy metabolism rates
• Katp channel – (proposed) to be involved in mitochondrial volume regulation
• PTP large pore – pathological channel involved in cell death
• NCX – (Sodium-calcium exchanger) helps to recycle calcium
• UCP – uncoupling protein – heat generation.
• VDAC – passage for metabolites across the outer membrane
• MAC – apoptosis channel
Investigating Mitochondrial Function
In most cases mitochondria
needs to be intact since integrity of the membrane is required
What can be measured in the intact mitochondria:
• NADH levels (imaging)
• Membrane potential (imaging)
• ATP levels (imaging)
• Oxygen consumption (respirometry)
-Place cells (mitochondria in air tight chamber) and
See how quickly oxygen is taken up (how the cells
Are “breathing”)
Flavonoid Quercetin
Addition of the flavonoid Quercetin to the neuronal culture leads to the increase of the mitochondrial membrane
• So the drugs that we believed improved mirochondrial function also helps
protect the brain.
• Flavonoids (E+Q) improve mitochondrial function by increasing the
spare respiratory capacity of the mitochondria
E+Q protect brain from stroke damage
Mitochondria and Pathology
• Cardiac cells heavily rely on ATP, but as soon as you depolarize the
mitochondria you will remove that membrane potential and ATP will
no longer be produced.
• Without ATP supply the cardiac cell is dead within a few minutes
Respirometry Example
(Initial control: rate they take up oxygen at first)
Then you add Oligomycin and it drops
then FCCP was added
And then Rotenone test and Antimycin test
* Oligomycin blocks ability of mitochondria to produce ATP (ATP synthase)
* FCCP resents membrane potential (does not allow a gradient to be formed) leads to increase in Oxygen because the only step that is occurring is the oxidation NO PHOSPHORALIZATION
* Rotenone stops complex I
* Antimycin stops complex III
Mitochondrial function during Stress
• What happens during stress, is that you have a lot of influx of calcium. Stress is for example, in case of heart attack, lack of oxygen. So mitochondria cannot function well. You have this gigantic influx of calcium which enters the mitochondria.
• This leads to the phenomenon called calcium induced permeability transition pore. Which is condition that occurs during cell death, stroke, or
heart attack.
- This porin resets membrane potential. The pore will open, it will depolarize and will cause massive tissue damage.
- This pathological pore which opens and kills mitochondria. Apparently what is important about it is that if you block this channel during this condition like in stroke you can significantly protect and diminish the tissue damage.
**• PTP – Permeability Transition Pore opens during calcium overload; leads to the loss of the membrane potential****
Mitochondria control the programmed cell death – apoptosis
- mitochondrial intermembrane space is not only space for energy metabolites but also space for pro apoptotic molecules like Cytochrome C, AIF; SMAC/DIABLO molecules
- They are stored there and are harmless for the cell unless apoptosis signal comes
• Then apoptosis signal leads to the formation of the mitochondrial apoptosis channel (MAC), which is large pore. Even larger than your traditional pore. The channel leads to the leak of these molecules into the
cytoplasm which trigger apoptosis.
Peroxisomes
- Membrane bound organelles (single membrane)
- Break down alcohol; toxins and fatty acids
• Note the outcome of the fatty acid hydrolysis is production of energy rich molecules of FADH2 and NADH (same enzymes as the mitochondria are
producing the same energy rich molecules except they are breaking down different things)
Cytosol components
- Water (85%) = semi-gel
- Proteins
- Carbohydrates
- Lipids
- Nucleic acids
- Buffers
- Ions
- Trace elements
Cytosolic and Cytoskeletal Functions
Maintenance of cell shape
- Every cell in the body or different types of cells in the body all have a different shape. That maintenance of shape comes from the cytoskeleton.
Whole cell movement
- Not all cells just sit there. Many of them move around. The ones that are moving around a lot are the white blood cells. They function usually in
connective tissue not blood vessels.
Contraction
Changes in cell shape
-There is a lot of places where cells can change shape and its important for
them to change shape. Somewhat during development.
Structural integrity of cell
-The cytoskeleton places an important role in structural integrity; in many cases, especially the intermediate filaments.
Organization and movement of organelles:
- mitochondria and the secretory granules and other organelles are organized and they move because of the cytoskeleton on the inside.
Cytosol Components
three types of filaments in the cytosol
• Mircofilaments – they are the smallest, 7-8 nm (will not ask numbers on the exam)
- Microtubules – largest filaments, about 24 nm
- Intermediate filaments – as electron microscopy got better, they discovered an intermediate filament
• Plus accessory and regulatory proteins of each are also part of the cytoskeleton
Actin
• It’s the component of MICROFILAMENTS
• It’s globular protein
• The monomer of the globular actin is G ACTIN, it will polymerize to form a filamentous chain which is called F-ACTIN
>The polymerization requires ATP and Magnesium.
> Microtubules and microfilaments have a polarity to
them. They form from a minus end towards the
plus end.
>If they’re not capped or maintained in some way,
they disassemble. The disassembly takes place
from the minus end of the filament in actin filament.
> Bundles of F-actin are referred as microfilaments.
> It’s really F-actin that is stabilized, it’s capped on
both ends so that it’s not continuing to
disassemble or to fall apart. It plays a role in
whatever the function of that cell might be.
• Different tissue in the body have different isoforms of actin. They’re basically the same but they have physiology functions that vary depending
on the isoforms of actin.
Different isoforms of actin (not going to talk a whole lot about these)
- α-skeletal - skeletal muscle
- α-cardiac - cardiac muscle
- α-vascular - vascular smooth muscle
- γ-enteric - visceral smooth muscle
- β-cytoplasmic - non-muscle cells
- γ-cytoplasmic - non-muscle cells
Muscle Cells
actin filaments are anchored in a very
regimented way in skeletal or cardiac muscle into these areas called the Z DISC
They interact with bundles of another cytoskeletal protein, MYOSIN
They induce a sliding of the filament. The sliding filaments take place in the non-muscle cells as much as they do in the muscle cells.
Non-Muscle Cells
They play the role in function related to cell shape and motility.
- Most of the functions in the non-muscle cells involve attachment to the cell membrane – they do that through interacting with proteins that are transmembrane proteins in the cell membrane.
- There is a bunch of actin binding proteins that are necessary for the actin to function within the cell.
Actin Binding Proteins
- Tropomyosin is important to skeletal and cardiac muscle.
- Alpha actin is important in muscle and non-muscle cells. These two are important in bundling filaments and something called microvilli.
- Spectrin is very important in crosslinking the membrane skeleton and something that is called terminal web.
- Myosin is important in sliding filaments in muscle and non-muscle cells.
- There are various Caps, the various capping proteins keep the filaments from depolarizing.
Actin Filaments function in Cell Shape Change
( 4 examples)
- Epithilial cell – are polarized, have a base and an apex, they can have cell surface and lateral specializations, can have finger-like projections called MICROVILI. They increase cell surface area. So you really see that these cells are involved in absorption. They absorb area of the GI tract (small intestine mostly as well as salivary gland ducts and kidney)
- Stress fibers - They position organelles and move organelles in very distinct patterns within the cell
• Cell motility – lamellipodium forms and you polymerize actin. Actin
myosin interaction then pulls up the rear end of the cell leaving bits and pieces of cell membrane and extracellular matrix and maybe integrins behind.
• Actin forms the contractile ring that separates the two daughter cells during Mitosis
Cell Motility
Cells form these LAMELLIPODIUM/Filapodia.
As the lamellipodium forms and you polymerize actin, it protrudes the front of the cell out further and further. It forms, because there are actin filaments in there and they are binding to integrins on the cell membrane, those integrins then bind to the extracellular matrix.
Cells don’t get infinitely long and skinny. What ends up happening is that the rear part of the cell has a actin
myosin interaction and it PULLS UP the rear end of the cell leaving bits and pieces of cell membrane and extracellular matrix and maybe integrins behind.
Contractile Ring in Mitosis
When cells go through mitosis and they divide, actin forms the contractile ring that separates the two daughter cells.
The contractile ring and the cleavage furrow is very important actin component in mitosis. Microtubules play a role in chromosome movement and separation.
Actin Attachment in Cell Membrane
Actin has to attach to the cell membrane to effectuate any of those sorts of cell shape changes.
ACTIN FILAMENTS are bundled together by protein ALPHA ACTININ
It comes through an ADAPTER COMPLEX that is made up of VINCULIN, and TALIN and a bunch of other specific proteins. They BIND TO A TRANSMEMBRANE
RECEPTORS. In this case it’s the INTEGRINS. Those integrins either bind to the EXTRACELLULAR MATRIX and something called the FOCAL ADHESION.
Actin can also bind to the same structures embedded into the membrane. As the actin interacts with myosin and it contracts, it can cause a contractile ring.
Terminal Web
In RBC, the terminal web includes GLYCOPROTEINS
There is a series of proteins that are very well studied ANCHORING BAND 3, PROTEIN 4.1, that organize this
altogether. What binds to ANKYRIN in this is transmembrane complex is a protein called SPECTRIN.
Spectrin is a super family. Spectrin binds to the
transmembrane protein at various spots all around this cell. ACTIN and protein 4.1 help crosslink and bind these together as a certain amount of tension is put on the system that gives you this biconcave disc. Every cell has a TERMINAL WEB.
Spectrin Gene Superfamily
- Spectrin I is in the erthrocytes (RBC) only
- Spectrin II is in every other cell in the body just underneath the cell membrane in various spots.
- Alpha Actinin - going to see it in skeletal muscle, see it in cardiac muscle and other muscles.
- Dystrophin – found in skeletal muscles. It is called dystrophin because it was discovered, in mostly boys at the time, that had duchenne and becker muscular dystrophy. Dystrophin was missing. Muscular weakness results and eventually death because even diaphragm doesn’t work.
Microvilli
Bundled actin filament have a structural function within microvilli
- Actin bundles are attached at their plus ends to the tip of the microvilli
- The actin filaments are bundled by the proteins VILLIN and FIMBRIN
- The actin bundles are attached to the sides walls of the microvilli plasma membrane via myosin I and calmodulin
- Within the terminal web, nonerythroid specturin (SPECTRIN II) and MYOSIN II links adjacent actin bundles to eachother and to intermediate filaments.
Microtubules**
MICROTUBULES
- Microtubules are made up of monomers or dimers of alpha and beta tubulin. Independent gene family products.
- They require GTP to polymerize.
- They assemble from a minus end, which is where things tend to disassemble.
- They have a growing end which is the plus end.
- The minus is always bound at something called microtubule organizing center and there are bunch of them inside cells.
• When the microtubules start to form, they start to form at an organizing center which is where the minus end tends to attach. It require GTP and the
GTP is on the terminal dimer
- But if you hydrolyze or dephosphorylate the terminal dimer, it leads to something called dynamic instability and the microtubule collaspes
- Microtubules continually grow from the centrosome, added to a cell extract. Quite suddenly however, some microtubules stop growing and then shrink back rapidly. A behavior called DYNAMIC INSTABILITY.
- Microtubules are the 13 protofilaments.
• Outside diameter is about 24 nanometers
.
• Inside, it is really a hollow tubule, its about 14 nanomenters.
• You add them on beta to alpha. They’re added on at the plus end and they peel off at the minus end unless you dephosphorylate these at the terminal end then the microtubules will collapse.
Microtubules and Cell Division
In the cell they all tend to startout at something called the CENTROSOME = when two centrioles line up perpendicular to each other.
Then there is a cloud of associated proteins that from these things called microtubule organizing center, these microtubules pull chromsomes apart
during cell division.
There are other microtubules organizing center. The one that is very well known and studied is the CILIA (long projections off the cell surface) in epithilial cells.
They form off of the basal body which binds the negative end.
Cilia function to move material across the cell surface. (EX. particles trapped in mucous on the surface of the trachea and lung)
Cilia
Cilia have a power stroke that takes place when the cilia are extended up into the mucus and moving into the direction they are organized to.
They drop off the mucus down into the liquid layer, recoil back, come up again around, hit the mucus and move it forward. They do this very fast and in an
organized fashion.
CILIA DO NOT MOVE WHEREAS FLAGELLA DO MOVE ( one of the major
differences between the two)
Centrosome
I. Contains two centrioles
II. Centrioles composed of 9 triplets of microtubules in a circle
III. Centrioles are perpendicular to each other
IV. Surrounded by a cloud of microtubule binding proteins (microtubule organizing center that binds minus ends)
Microtubule Movement
Vesicles in microtubules and there are a couple of different motors.
There is a motor that moves organelles from the minus end of the microtubule to the positive end. That motor protein called KINESIN.
There are those that move from positive
end back to where the centrosome is or cell center called DYNEIN.
• Where this is really magnified is in an axon on a motor neuron where these motor proteins dragging things
• dynein moves things towards the minus end (towards the cell center)
• kinesin goes towards the plus end (periphery of the cell)
Intermediate Filaments
Intermediate filaments are much more structural – are holding the cell into its shape.
They don’t move things, they don’t have motor proteins associated, they don’t polymerize.
- Intermediate filaments have a number of gene products.
- The proteins in every type of cell are different but the general function is the same.
- EPETHIAL cells have keratins.
- The gene family which is the vimentin and vimentin related genes are in CONNECTIVE TISSUES
- Then there is a special class in nerves called neurofilaments in NERVE cells.
- So although there are different proteins in different tissues, the general structure is the same.
- It’s an alpha helical monomer, it’s got an amino terminus and a carboxy terminus.
It forms a coiled dimer that work together»_space;> forms
tetramer»_space;» form two tetramers »_space;» they pack together putting 8 tetramers together »_space;> that gets coiled into a rope like filament that is 10 nm across.
- It’s a very structurally rigid, strong, filament.
- Does not do dynamic instability like microtubules do. They don’t spontaneously disassemble if you remove a regulatory protein.
- Intermediate filaments attach to desmisomes and hemidesmisomes
Neuroglia
Cellular connective tissue of the CNS
Astrocytes (Astroglia)
- Pure connective tissue cells - Vimentin and GFAP
Oligodendrocytes (Oligodendroglia)
- Contractile and produce myelin
- Vimentin only
Microglia (Mesoglia)
- Macrophages - Vimentin only
Dynamic Instability
Microtubules are highly dynamic and will frequently grow and shrink at a rapid yet constant rate. During this phenomenon, known as ‘dynamic instability’, tubulin subunits will both associate and dissociate from the plus end of the protofilament
the primary determinant of whether microtubules grow or shrink is the rate of GTP hydrolysis,
