Lecture 2: Cell structure and function Flashcards
What can be seen with the unaided human eye?
tick, human heart, dog
What can be seen with the compound light microscope?
Chlamydia, rickettsia, mitochondrion, bacteria, chloroplast, RBC, large protozoa, human egg, tick
What can be seen with the scanning electron microscope?
Ribosomes, viruses, chlamydia, rickettsia, mitochondrion, bacteria, chloroplast, RBC, Large protozoa, human egg, tick
What can be seen with a transmission electron microscope?
AA’s, diameter of DNA, proteins, ribosomes, viruses, chlamydia, rickettsia, mitochondrion, bacteria, chloroplast, RBC, large protozoa, human egg
Large protozoa
eukaryotic microbe
- has euk cell structure
- larger than a prok, but smaller than a plant
- light microscope
Red blood cells
- small because they need to single file in the capillaries
- at maturity they lose mitochondria and all organelles allowing them to be small
Chloroplast
- organelle
- according to endosymbiosis theory they were prokaryotes
Chlamydia
Bacteria
- OBLIGATELY INTRACELLULAR
- smaller than bacteria (bc cell provides a lot for it)
- doesn’t juts live free in the reproductive tract but goes into reproductive tract when you 1st get the infection and then into the cytoplasm of the cell where it grows
What is similar between chlamydia and viruses?
- both organisms that go into eukaryotic cells and cause infection
- obligate intracellular
BUT
- chlamydia still have ribosomes but viruses have none of that making them smaller
Why is chlamydia small?
anything that lives inside of a cell loses what it does not need bc cell provides it a lot
But it still will have ribosomes etc stuff which viruses wont have
What should you be given if you have chlamydia?
The Dr needs to choose antibiotics that don’t just go into intracellular fluid but that go into cytoplasm of the cell
- antibiotic goes from (blood –> tissue –> ECF –> cytoplasm to find target
- antibiotic is said to have good tissue penetration
- if you choose wrong antibiotic your not helping clear the inefction
If someone had chlamydia & got a swab. After its been spread on petri dish & incubated, will there be chlamydia growth the next day?
NO IN A LAB SETTING WE CANT GROW IT
- we haven’t been able to figure out the conditions to grow chlamydia
- they have to diagnose it by nucleic acid testing
rickettsia
an INTRACELLULAR ORGANISM
- ancestor of mitochondria
- goes into a cell
- may have got trapped to act like a mitochondria (replicated)
Compound light microscope
uses visible light to illuminate the sample
- just a light bulb is used which is a low energy source so there’s limitations
What are the many different types of light microscopy?
- Bright-field
- Phase-contrast
- Dark-field
- Fluorescence
Bright-field scope
• Specimens are visualized because of differences in contrast between specimen and surroundings (dark cells on bright background)
• Two sets of lenses form the image
• Objective lens (usually 10x -100x mag.)
& ocular lens (usually 10x – 20x mag.)
Maximum magnification is ~2,000✕
Total magnification
objective
magnification ✕ ocular magnification
Why do we call the bright-field scope a compound light microscope?
b/c it compounds the magn. that the 1st lens is giving you by further magnifying it through a 2nd lens
Condenser
creates a beam of light so it’s condensed/focused to be able to move through the microscope slide
Describe the magnification light path
- Light from light source
- Condenser - focuses it into a beam so it’s interacting with the specimen
- Once the specimen comes through the objective lens (10X, 40X, or 100X (oil)), it’s inverted in position & magnified to whatever you chose
- When the specimen comes through the ocular lens (10X) it is inverted again to OG position & magnified further, so even larger
- Then you see it at either 100X, 400X, 1000X
Magnification
the ability to make an object larger
Resolution
the ability to distinguish two adjacent objects as separate and distinct
• Limit of resolution for light microscope is about 0.2 μm
(light can only get thru spaces that are at least 0.2 μm)
minimum distance 2 objects need to be apart in order for microscope to show those 2 objects
-> if objects are LESS than minimum distance that you see a blurry image
If we had a better microscope, what would we expect the limit of resolution value to be?
SMALLER - b/c then those 2 objects can be even closer together & you can still see a clear image (ex: e- microscopes)
What is relationship between wavelength and energy?
INVERSELY PROPORTIONAL
- as one increases the other decreases
Resolution explained
- The ability of a lens to distinguish small objects that are close together
- Ex) resolving power of 0.2μm
- Two points can be distinguished if they are at least 0.2 μm apart
- Light must pass between two points for them to be viewed as separate objects (providing clarity)
What happens as wavelength decrease?
resolution improves
Longer wavelength=
↓ ENERGY
↓ RESOLUTION
Shorter wavelength =
↑ ENERGY
↑ RESOLUTION
Why is increased energy and shorter wavelengths dangerous?
DANGEROUS b/c energy it carries can be transferred all the way to DNA for ex & can result in damage/mutation that can lead to cancer
- ↑ energy radiation is dangerous b/c energy it carries will then be transferred to other objects its in contact with
↓ energy - longer wavelengths =
safer
If you have 2 microscopes, both with the same magnification, but diff. resolving powers. Will they provide the same image?
NO - bc as you magnify, it doesn’t mean the resolution maintains clarity
- ex: zooming on phone doesn’t mean it will still be clear
GREATER magnification…
DOESN’T mean/guarantee resolution will also increase; dependent on microscope you chose
Throw ink-covered objects at target (“E”):
- Basketballs - longest wavelength
- Tennis balls - slightly shorter wavelength
- Jelly beans
- Beads - shortest wavelength
- Cannot fit between arms, poor resolution
- Fit between arms, resolution improves
3 and 4. As diameter of objects thrown decreases, greater numbers pass between the arms & the resolution increases
What improves contrast what does this result in?
Improving contrast results in a better final image
• Staining improves contrast
How does staining improve contrast?
Dyes are organic compounds (carbon containing CH2-COO-) that bind to specific cellular materials (inside the cell)
What is resolution based on?
energy source
Name some common stains
methylene blue, safranin, and crystal violet
Simple staining
One dye used to color specimen
- just shows if something is THERE or NOT
“one size fits all”
Chromophore
colored portion of a dye
- colour your cell will appear as a conseq. of that dye adhering to portions of the cell or cellular structures
If a basic blue chromophore is used what colour will the cell be?
basic= +
cell is negatively charged so they attract and cell will be blue
Basic dye
positively charged chromophore
• Binds to negatively charged molecules on cell
surface
- (+) at pH=7
ex- crystal violet
What are the 2 types of simple stains/dyes?
- Basic dye
2. Acidic dye
A living cell, whether it’s a bacterial cell, fungal cell or human cell, will….
ALWAYS HAVE A NET (-) CHARGE
- if you apply a basic stain it will adhere
- if you apply an acidic stain it will repel
Acidic dye
negatively charged chromophore • Repelled by cell surface • Used to stain background • Negative stain (-) at pH=7
ex- nigrosin
How to prepare samples for staining
- Preparing a smear
Spread culture in thin film over slide
Dry in air - Heat fixing and staining (dehydrating sample so it is stuck on slide, therefore it doesn’t get rinsed off in next step)
- Pass slide through flame to heat fix
- Flood slide with stain; rinse and dry - Microscopy
- Place drop of oil on slide; examine with 100
objective lens
- NO DIFFERENTIATION, just can see that there’s pink circles
- details on cell shape size arrangement
Gram - & gram + cells will BOTH…
be negatively charged on their external surface
What is the difference between gram + & gram -?
architecture of their cell wall is different
The Gram Stain
a differential stain
Separates bacteria into 2 groups based on cell wall structure
What is a reason to perform the gram stain?
It is useful to know if organism is gram + or - bc some antibiotics target only certain + or - so its useful to know
If someone has a gram + infection
you take antibiotics that target gram + bacteria you leave the gram - bugs alone
- better toxicity
Gram positive
cells that retain a primary stain
• Purple
Gram negative
cells that
lose the primary stain
• Take color of counterstain
• Red or pink
Gram + general features
- plasma membrane
- THICK peptidoglycan
Gram - general features
- plasma membrane
- THIN peptidoglycan layer
- outer membrane
What is the Gram Stain procedure?
- Apply CRYSTAL VIOLET stain purple & basic:
Gram + = purple
Gram - = purple
Human cell = purple - Apply IODINE = a mordant - intensifies bound stain
-> bulkens up the stain molecules so they will stay trapped
Gram + = purple
Gram - = purple
Human cell = purple - Apply ALCOHOL = a decolourizer
Gram + = purple (trapped in cage like structure - alcohol bulked them up so they can’t get mordant & crystal violet out)
Gram - = colourless
Human cell = colourless (bc no cell wall) - Apply SAFRANIN (pink & basic) - sticks to any living cell
Gram + = purple (pink will stick but will stay purple b/c it never came off & its darker)
Gram - = pink
Human cell = pink
Gram stain but everything is mixed up
If in step 1 of this gram staining protocol, instead of crystal violet, a yellow basic stain is added & in step 4 instead of safranin, a black basic stain is added, what will be the outcome for that gram stain result/ For a gram +, - & euk?
everything will be BLACK bc the darker one was added after alcohol so it will trump yellow bc darker
Explain why gram negative cells appear pink in the gram stain?
gram negative have outer membrane so when you douce in crystal violet it will stick bc the cell has a net negative charges but when alcohol is added it will remove colour
Acid fast stain
differential stains
Detects mycolic acid in the cell wall of the genus Mycobacterium
- Mycobacterium – retains primary stain
• Fuchsia (pink)- carbol fusion sticks to mycolic acid
Anything else on slide – color of counterstain
• Blue
Mycobacterium genus
has plasma membrane, gram +, & MYCOLIC ACID (hydrophobic) as the outside of their peptidoglycan
Mycolic acid
- hydrophobic
- outside of their peptidoglycan
- unique to members of mycobacterium genus
- can engage with things ALSO hydrophobic
Mycobacterium CANNOT…
undergo a gram stain
- so has to do AFS
Will mycobacterium be able to engage with a basic or an acidic stain?
No
bc like dissolves like so they will engage with hydrophobic things
acidic and basic stains are charged meaning they are hydrophilic so these stains won’t work
What would be a positive result in a acid fast stain?
pink
- which shows mycobacterium everything else, all other cell types are blue
Methylene blue (basic stain) AFS
basic= positive charges
so it’ll stick to cells that aren’t acid fast
-> stick to other gram +/- , human cells, anything that has a net negative charge on outermost surface
What would be a negative result in an acid fast stain?
EVERYTHING is blue nothing is pink so you can perform a gram stain for further analysis
If a Dr suspects mycobacterium what should they do?
they want to collect enough of a sample for 2 tests
1) to do acid fast stain
2) gram stain
When you gram stain on a gram + bacterium, what’s the expected outcome?
the stain to remain purple b/c it’s trapped in peptidoglycan, it won’t come out (not outer membrane)
Endospore stain
Endospores retain primary • Green • Cells counterstained • Pink- safranin • Ex. Bacillus anthracis spores.
What are endospores?
*PRODUCED BY ONLY SOME BACTERIA AND ALWAYS PRODUCED BY GRAM POSITIVE BACTERIUM
resilient structures
ex: if a bacterium finds themselves in a situation where water/nutrients are limited, they can enter in spore state (metabolically inactive) where he hides out until conditions become more favourable for survival, then he comes out & wakes up & starts metabolism & being normal again
ex: rice
- when they fuel replication from those conditions, the microbial count goes up!
- & if you take it & put it on fridge, it will grow slowly
- can destroy with an autoclave- combines temp & pressure for ex
How would you expect a (-) endospore stain to look?
Everything would be pink
If everything here was pink (- endospore stain), would you be able to tell me what the result of a gram stain is?
NO- b/c you’ll have bugs there that the pink is sticking too but you don’t know if its gram + or - (it can be either)
- it’s only when you know it produces endospores, that you know it must be gram + & therefore will produce a purple stain
If you chose this endospore forming organism, but instead chose to do a gram stain. What would I see as a result of the gram stain on an organism that produces a (+) endospore stain?
purple - because it’s an endospore forming & gram + are the only kind of bug that’ll form an endospore
Phase-contrast microscopy
Phase ring amplifies differences in the refractive index of cell and
surroundings
• Improves the contrast of a sample without the use of a stain
• Allows for the visualization of live samples
• Resulting image is dark cells on a light background (similar to general microscope but not pretty colours)
Dark field microscopy
Specimen is illuminated with a hollow cone of
light
• Only refracted light enters the objective
• Specimen appears as a bright object on a dark background
• Used to observe bacteria that don’t stain well
• Ex) Treponema pallidum – the causative agent of syphilis
opposite of phase contrast
Fluorescence microscopy
• Used to visualize specimens that fluoresce
• Emit light of one color when illuminated with another color of light
• Cells may fluoresce naturally
• Ex. Photosynthetic Cyanobacteria (origin of eukaryotic chloroplast) have
chlorophyll
• Absorbs light at 430 nm (blue-violet)
• Emits at 670 nm (red)
• Or after staining with Fluorescent dye
• Ex) DAPI specifically binds to DNA
absorption wavelength
characteristic of the chemical properties of a fluorescent particle
- have to know what this is so it can be absorbed
- have to strike this with light of a particular wavelength
emission wavelength
what’s given off to provide the fluorescents that you see
If absorption wavelength is 450 nm, what can you say about the emission wavelength?
LARGER wavelength - because of the energy that was absorbed, some of it was converted to heat - so not all of it will be there
- b/c there is a loss of heat, when this gets absorbed you’re gonna have less that’ll come out which means that wavelength must be larger b/c larger wavelength is lower energy which counts for amount spent in absorption activity
Immunofluorescence
take antibody and couple it with fluorescent particle then put it into cell and treat the cell with absorptions wavelength
Differential interference contrast (DIC) microscopy
- Uses a polarizer to create two distinct beams of polarized light
- Gives structures such as endospores, vacuoles, and granules a three- dimensional appearance
- Structures not visible by bright-field microscopy are sometimes visible by DIC
- still using light microscope but structures not visualized by brightfield can be seen by this
Confocal scanning laser microscopy (CSLM)
- Uses a computerized microscope coupled with a laser source to generate a three- dimensional image
- Computer can focus the laser on single layers of the specimen
- Different layers can then be compiled for a three-dimensional image
- Resolution is 0.1 μm for CSLM
three dimensional layering
Electron microscopes
use electrons instead of photons to image cells and structures
• Wavelength of electrons is much shorter than light –> higher resolution
nm ranges in resolution
reverse direction of light microscope
What are the 2 types of electron microscopes
• Transmission electron microscopes
(TEM)
• Scanning electron microscopes (SEM)
Transmission Electron Microscope
Electron beam focused on specimen by a condenser
• Magnets used as lenses
• Electrons that pass through the specimen are focused by two sets of lenses
• Compound microscope
• Electrons strike a fluorescent viewing screen
High magnification and resolution (0.2 nm)
Specimen must be very thin (20 – 60 nm) (Inject it with resonance so that cell hardens, creating a solid structure)
use microtome= diamond knife to do thin sectioning
*USED TO SEE OUTSIDE OF THE CELL
Unstained cells do a poor job of scattering electrons
Must be stained with metalsa=> lead or uranium
=> metal is heavy means subatomic particle number will be high= e- number is high
=> heavy metal stain will be electron dense will stick differently that to ribosome or DNA
• Bind to cell structures to make them more electron dense
• Enables visualization of structures at molecular level
Difference in Resolution Between Light and Transmission Electron Microscope
magnification is constant but EM provides way better resolution
- EM has much smaller resolving power
Scanning Electron Microscopy (SEM)
• Specimen is coated with a thin film of heavy metal (e.g., gold)
• An electron beam scans the object (rather than going thru thin section
• Scattered electrons are collected by a detector, and an image is
produced
• Allows an accurate 3D image of specimen’s surface.
*SEE OUTSIDE OF THE CELL
Prokaryotes
(before nucleus)
• No membrane bound nucleus or organelles (genetic material is floating around cell)
• Generally smaller than eukaryotes (bc they pack alot less)
• Simple internal structure
• Divide by binary fission (get whole new organism rather than in us where we increase total concentration of cells)
• Most are unicellular
What is binary fission equivalent to?
Mitosis bc resulting in genetically identical daughter cells no genetic variation just a means of increasing cell number when we do mitosis we increase in cell number
binary fission is ur getting a whole new organism
Bacteria (Eubacteria)
- Diverse metabolism (allows them to live in diff environments)
- Live in a broad range of ecosystems
- Pathogens and non-pathogens
Archaea (Archaebacteria)
- Diverse metabolism
- Live in extreme environments
- Non-pathogens
- only thing they share with bacteria is prokaryotic structure
Coccus (pl. cocci)
• Roughly spherical • Ex) Streptococcus pyogenes - causes strep throat - flesh eating bacteria *same species but different strains
Bacillus (pl. bacilli)
• Rod shaped
• Ex) E. coli
- bladder infection, fecal contamination
- asymmetrical shape
Spirillum (pl. spirilla)
- Spiral shaped
- Ex) Spirillum volutans
- helical morphology
- shorter length, rigid causes restricted flexibility
Cells with unusual shapes
• Spirochete
• Ex) Treponema pallidum
- helical confirmation but longer so more flexible
- STI
Budding & appendaged bacteria (allows it the ability to make contact with the surface)
• Ex) Caulobacter crescentus
Filamentous bacteria
• Ex) Streptomyces griseus
- long and thin so good SA for absorption
Morphology typically does not predict
physiology, ecology, phylogeny, etc. of a prokaryotic cell
has nothing to do with genetics either
What are the selective forces that may be involved in setting the morphology?
• Optimization for nutrient uptake (small cells and those with high surface-to-
volume ratio)
- sa; v ratio- better membrane to interact with enviroment
• Swimming motility in viscous environments or near surfaces (helical or spiral-shaped cells)
- if cell has particular shape it can cut thru and move one
• Gliding motility (filamentous bacteria)
- maximize contact with environment
Describe the different cell arrangements
diplococcus
diplobacillius
streptococci
staphylococci
Prokaryote Size
- E.coli~1.0x3.0μm
- Staphylococcus aureus ~ 1.0 μm diameter
Very small:
• Mycoplasma genitalium ~ 0.3 μm (STI)
-> anmaly bc it doesn’t have a cell wall
-> outer most component is the plasma membrane similar to a animal cell which doesn’t have a cell wall so you would not put into a gram stain bc it would look pink bc alchol would destain the membrane so gram stain is not an effective way to identify
Very large:
• Epulopiscium fishelsonii ~ 80 x 600 μm.
Cell Size and the Significance of Being Small
Surface-to-volume ratios, growth rates, and evolution
• Advantages to being small
• Small cells have more surface area relative to cell volume than large cells (i.e., higher S/V)
• Support Greater Nutrient Exchange Per unit cell volume
• Tend to grow faster than larger cells
Lower limits of cell size
• Cellular organisms <0.15 μm in diameter are unlikely
• Open oceans tend to contain small cells (0.2–0.4 μm in diameter)
• Many pathogenic bacteria are also small (missing many genes whose functions are supplied to them by host)
- makes things more efficient
- you have to fit ribosomes etc in so they can only be so small
Describe the Cytoplasmic membrane (cell or plasma membrane)
• Thin structure that surrounds the cell
->makes it easier for nutrients to come in and waste to leave
- Vital barrier that separates cytoplasm from environment
- Highly selective permeable barrier; enables concentration of specific metabolites and excretion of waste products
- > chooses what gets in
- > cell has the ability to let glc and lactose in
- > wastes can be toxic if they accumulate so its imp part of membrane function to let wastes out
*BOTH PROKARYOTES AND EUKARYOTES HAVE THIS
Do viruses have a cytoplasmic membrane
NO
Describe the composition of membranes?
- General structure is phospholipid bilayer (2 layers)
- Contain both hydrophobic (fatty acid) and hydrophilic (glycerol-phosphate) components
- Can exist in many different chemical forms as a result of variation in the groups attached to the glycerol backbone
- Fatty acids point inward to form hydrophobic environment; hydrophilic portions remain exposed to external environment or the cytoplasm
Amphipathic
membrane is amphipathic because it has both polar and non polar components
- polar heads non polar tails
Describe the glycerol backbone & attachments
- 3 carbon back bone
c1; has x group which determine identity
c2 and c3; have fA tails which determine characteristics depending on length or saturation vs unsaturation
How is the phospholipid bilayer based on the Hydrophobic Effect?
According to the hydrophobic effect if you put a phospholipids into water they naturally form a bilayer in order to form favorable interactions
What would happen if you put phospholipids into a beaker of oil?
The hydrophobic tails will interact favourably with the oil (nonpolar with nonpolar)
and the heads will face inward
What do the exposed tails do?
they come together and naturally circulize
-> ends come together so they aren’t touching water
Unsaturation vs saturation
Unsaturation=> lower boiling point, more fluidity, less vdws so less what required
saturation=> higher boiling point, melting point, solid at room temp, more vdws so more heat to break
Ester phospholipids consist of:
glycerol ( 3 carbon backbone with 3 OH groups)
• 2 Fatty acids
• Phosphate
• Side chain (optional)
Polar:
molecule carries full or partial charge
• Hydrophilic
Non-polar
molecule is
uncharged
• Hydrophobic
electrons are held in the middle- non polar covalent bond
What can we say about the bonds between N & H in +NH3?
the electrons are held on N more closely bc polar covalent bond
What is the charge on the membrane and why?
The membrane has a net negative charge
- look at picture in notes
- the x group charge can make it have net negative charge
Cytoplasmic membrane
- 8–10 nm wide
- Embedded proteins
- Stabilized by hydrogen bonds and hydrophobic interactions
- Mg2+ and Ca2+ help stabilize membrane by forming ionic bonds with negative charges on the phospholipids
- Somewhat fluid
What particularly will be determining width of membrane?
Length of FA tails
- > which determines how far the head will be from one another
- > if you adjusted length to much the protein wouldn’t insert properly into membrane
What’s the net charge on a membrane?
membrane has a net negative charge
How do membranes stay together so nicely if they have a compounded net (-) charge?
Mg2+ and Ca2+ help stabilize membrane by forming ionic bonds with negative charges on the phospholipids
- > membrane has net negative charge which means the phospholipids repel each other
- > divalent cations help with this
How does the membrane need to be in order to function?
semi fluid
- > if fully fluid like puddle of oil it wont work and if hard like rock that won’t work
- > The tails are far apart so you can’t get vdws interactions so it creates fluidity bc the tails are not locked in position
What amino acids will be in the interior of the membrane?
Non polar amino acids
-> uncharged hydrophobic
What bonds hold the membrane?
vdws interactions
-> weak interaction between non polar
Membrane proteins
- Outer surface of cytoplasmic membrane faces the environment
- In gram-negative bacteria, interacts with a variety of proteins (periplasmic proteins) that bind substrates or process large molecules for transport
- Inner surface of cytoplasmic membrane interacts with proteins involved in energy-yielding reactions and other important cellular functions
- Integral membrane proteins
- Peripheral membrane proteins
Integral membrane proteins
Firmly embedded in the membrane
-> analogous to inner mitochondrial membrane and will have ETC
Transmembrane proteins
- intergral membrane proteins
- go all the way thru membrane
Peripheral membrane proteins
One portion anchored in the membrane - are attached to extracellular face or intracellular - attache dto hydrophilic regions - using H bonds or ionic interactions (electrostatic interactions)
What type of linkages do Archaea have?
Ether linkages in phospholipids of Archaea
-. provides better thermal stability
What type of linkage do Bacteria and Eukarya have?
Bacteria and Eukarya that have ester linkages in phospholipids
Describe Archaeal membranes
Archaeal lipids lack fatty acids (REQUIRE CARBOXYLIC GROUP FOR FA THAT WOULD BE ESTER SO THEY DON’T HAVE); have isoprenes instead
• Major lipids are glycerol diethers and tetraethers
• Can exist as lipid monolayers, bilayers, or mixture
Isoprenes
C5 Tails- hydrophobic
- come together; c5+ c5= c10
Glycerol diether
2 ethers
- 1 glycerol neck
bilayer
Diglycerol tetraethers
2 glycerlo necks
4 ether linkages
- monolayer; no gaps meaning you get more vdws interactions so you have more to melt
What does more vdws mean?
membrane will hold better at higher temps
What Archaea membrane would they want @ 4 degrees celsius?
The bilayer- bc membrane will want to solidfy so you would want more of this to prevent bc these impart fluidity
Lipid bilayer vs monolayer
- monolayer; no gaps meaning you get more vdws interactions so you have more to melt
- bilayer- gaps so less thermal stability
What Archaea membrane would they want @ 60 degrees celsius?
Lipid monolayer
- impart a lot of stability
In contrast to lipid bilayers, lipid monolayer membranes are…
extremely heat resistant
- bc more vdws
Commonly found in hyperthermophilic Archaea (grow best at temperatures above 80oC)
What is able to pass through the lipid bilayer?
small non polar material can move freely along concentration gradient
Are polar things able to move freely across the membrane?
No
- are more controlled need transporters/ channels
- ex- pores for polar material which are very specific to a certain material
Membrane Function
Permeability barrier
• Polar and charged molecules must be transported
• Transport proteins accumulate solutes against the concentration gradient (ACTIVE TRANSPORT, moving things from areas of low to high bc they can’t freely pass thru membrane allowing it to accumulate)
Protein anchor
• Holds transport proteins in place
Energy conservation
• Generation of proton motive force
Do the proteins 1-4 have to stay in that position, or can they move?
they have to stay in that position bc its essential the ETC stays like this or you would lose function
If proteins were able to flow wherever they wanted b/c charge didn’t prevent that from happening, what would be the issue?
The gradient would disappear and energy could not be harvested so you want protons to move from high to low and stay so that ATP synathse can allow them to move thru and make ATP
Why is cyanide lethal?
toxins like cyanide make the membrane leaky to protons which means that the protons can mover wherever and don’t need to use ATP synthase so you lose the ability to harvest energy to make ATP and lose the ability to make gradients
this is lethal bc your energy production stops and brain run out of gas
Carrier-mediated transport systems
- Show saturation effect
* Highly specific (bringing in one particular material)
Simple diffusion
molecule is moving directly thru the PM (no transmembrane protein is required)
- very limited, inefficient bc molecule might be polar to large
Transporter
physically binding to and guiding molecule into cell rather than following the gradient
Explain the effect of a transmembrane protein (saturation effect)
transmembrane protein (polar)
- dramatic increase in solute entry and then levels off bc once all the binding sites are saturated it can’t go any faster
- > similar to enzyme activity; vmax cooks in the kitchen
What are Three major classes of transport systems in prokaryotes?
- Simple transport
- Group translocation
- ABC system
All require energy in some form, usually proton motive force or ATP (ALL ACTIVE TRANSPORT)
What do all 3 major classes of transport systems in prokaryotes require?
All require energy in some form, usually proton motive force or ATP
Explain simple transport
Driven by the energy in the proton motive force
- ex of secondary active transport
- > energy released from proton movement from high to low funds energy needed to move another solute from low to high
Explain Group translocation:
Chemical modification of the transported substance driven by phosphoenolpyruvate
For a cell that has an abundance of nutrients all around it, it will want to take in all these extra nutrients, so the cell responds by taking in much as possible and exceed equ’ and keep bringing in more by bringing it in and phosphorylating it your changing the molecule
when you do this its now a different molecule, so you wont reach equ’ between molecule and molecule phosphorylated
outcome is solute inside the cell is consistently staying low meaning you can bring lots in maximizing intake potential, a lot more can come in
Explain ABC transporter
Periplasmic binding proteins are involved and energy comes from ATP.
_ take ATP hydrolyze is and releases energy which is used to pay
-> type of active transport bc ATP is directly being spent
What are the 3 transport event possible?
uniport, symport, and antiport
Uniporters
transport in one direction across the membrane
Symporters
function as co-transporters - move in same direction
Antiporters
transport a molecule across the membrane while simultaneously transporting another molecule in the opposite direction
Co- transport
moving 2 things at once using one transporter
Lac permease of Escherichia coli
Simple transport
Lactose is transported into E.coli by the simple transporter lac permease
- a symporter- 2 molecules being moved in the same direction
- activity is energy driven
- transports H+ and lactose into the cell simultaneously
- proton releases energy which is used to pay for lactose movement
- lactose is going against concentration gradient, moving from low to high
phosphotransferase system in E. coli
Group Translocation
Sugar is phosphorylated during transport across the membrane
Moves glucose, fructose, and mannose
Phosphoenolpyruvate (PEP, is the most energy rich molecule used for substrate level phosphorylation) donates a P to a phosphorelay system
P is transferred through a series of carrier proteins and deposited onto the sugar as it is brought into the cell
*motivation is to try and establish equ but once equ is reached you can’t have more in so instead you phosphorylate it so its no longer glc no longer part of glc gradient so glc can keep coming in
ABC (ATP-binding cassette) transport systems
• Involved in uptake of organic compounds (e.g., sugars, amino acids),
inorganic nutrients (e.g., sulfate, phosphate), and trace metals
• Typically display high substrate specificity
• Gram-negatives employ periplasmic-binding proteins and ATP-driven transport proteins
• Gram-positives employ substrate-binding lipoproteins (anchored to external surface of cell membrane) and ATP-driven transport proteins
What does, “typically display high substrate specificity” mean for ABC transport?
you need many ABC in cell bc 1 is specific to one molecule; so a substantial amount of the bacterial genome is dedicated to providing instructions to make ABC bc you need a seperate one for each nutrient
Porin
non-specific transmembrane protein found in gram negative organisms
- is in outer membrane and is non specific as long as size and polarity is met
Periplasmic binding protein
found in gram negative organisms binds to nutrient and delivers it to ABC allowing the nutrient to be taken up by the cell
- you have these for nutrients stuff you want to come in not for toxins
ABC Transport is…
Describe ABC transport systems in a Gram - cell
porins in outer membrane (transmembrane protein)
-> nonspecific as long as polar and size is met then it can pass thru
> outer membrane is like a fence things can freely pass into the yard (periplasmic space) but to enter the house cytoplasm they need to go thru PM which is specific
enters periplasmic space where the nutrient float around
- periplasmic binding proteins are also floating around find nutrient they are specific to and bring to ABC transporter
Describe ABC transport systems in a Gram + cell
substrate binding lipoproteins
- lipoproteins are attached to PM, swimming around in extracellular fluid find nutrients for what its specific and guide it to ABC
ABC transporters (ATP-binding cassette)
Solute binding protein • Periplasm • Binds specific substrate Integral membrane proteins (transporter) ATP-hydrolyzing proteins • Supply energy for the transport event
