Membrane Permeability and Cell Compartments

Vocabulary practice flashcards covering cell membrane permeability, plasmolysis, Overton's rule, fluorescence microscopy optics, vital organelle stains, and cytoskeletal immunofluorescence.

Plasmolysis

The process that occurs when lots of water exits a cell through osmosis, causing the cell to shrink. In plant cells, this is easy to detect because the rigid cell wall makes the initial cell size visible.

Deplasmolysis

The process that occurs when lots of water re-enters a cell through osmosis, causing the cell to expand.

What happens when we put cells into hypotonic solutions?

Solute concentration is smaller outside, so water does NOT leave the cell, and plasmolysis does NOT occur

What happens when we put cells into a hypertonic solution?

• Solute concentration is bigger outside, so water leaves the cell and plasmolysis occurs.

• There is a lower (more negative) osmotic potential.

What’s the relationship between osmotic potential and solute concentration?

A measurement referring to the tendency of water to move across a membrane. As solute concentration increases, osmotic potential decreases (becomes more negative), driving water to move toward the solute.

Percent Plasmolysis

A quantitative measure of cell shrinkage calculated as: $\frac{\text{Change in protoplast size}}{\text{original cell size}} \times 100$

How did we estimate the total tonicity (total molarity of "stuff") in Elodea cells?

• By measuring the degree of plasmolysis, we could estimate the total solute concentration within a cell

  • Plotted sucrose concentration (M) vs plasmolysis percent (x axis)

  • We got the isotonic concentration of sucrose (where the solute concentration equals the stuff inside the cell) from the y intercept

Overton's Rule: How did we test it?

The rule stating that the ability of a solute to penetrate a membrane is directly related to that solute's lipid solubility.

Then we put three unknown substances on the elodea cells and used their relative percent plasmolysis and partition coefficients to determine which solution was which

How does the ether:water Partition Coefficient relate to how membrane permeable something is and plasmolysis?

• Bigger partition coefficient, more lipid soluble, more of that solute enters cell, less water out, less plasmolysis

• Smaller partition coefficient, less lipid soluble, more of that solute stays outside, more water out, more plasmolysis

Why do we sometimes see an initial plasmolysis, followed by a deplasmolysis all in the same solution?

• Water initially moves out, especially when cells put in solvent with smaller partition coefficients

• As time goes by, the inside becomes hypertonic because so many other cellular components AND some of that solute of the solution ends up coming inside the cell too slowly. This causes water to flow back in

Why was plasmolysis/deplasmolysis “too fast to observe”?

Bigger partition coefficient → solute crosses membrane super fast, so plasmolysis doesn’t happen and deplasmolysis doesn't happen as a result

Why are the stains we used in part I so vital for scientists?

Non-toxic (or low-toxicity) stains with relatively high partition coefficients that enter living cells without killing or fixing them, maintains the morphological relationships of subcellular components; specific

What’s a fluor and how does it work?

• A molecule/protein that is capable of undergoing fluorescence

• It gets excited by a specific wavelength of light, and as a result, it can emit a different specific wavelength of light

How does fluorescence microscopy work?

• The excitation wavelength goes onto the fluor and excites the electron of the outer shell

• The electron then goes back to the ground state and emits a new photon of light of lower energy

• In the actual microscope:

• 1. A light source shines all the wavelengths of light onto an excitation filter

• 2. The excitation filter then filters out the specific excitation wavelength

• 3. The excitation wavelength reaches the dichroic, which then directs the excitation wavelength to the specimen. 

• 4. The specimen then emits a new wavelength of light, which travels down through the emission filter and to the camera of the scope. 

Hoecht Stain

binds to the minor groove of DNA; it is excited by UV light, emits blue light, and stains cell nuclei appearing as large bright blue dots at the center of the cell

Bodipy Ceramide

in through endocytosis and shuttled to Golgi apparatus; ceramide “metabolite” that localizes to Golgi; Bodipy = fluor excited by blue light and emits green light

MitoTracker Green

linking to thiol groups in proteins with cysteine in the mitochondria; uses membrane potential of mitochondria to label; excited by blue and emits green

ER-Tracker Green

binds to sulfonylurea receptors of ATP-sensitive K+ channels in the ER; excited by blue light and emits green light; displays as a spider-web pattern throughout the cell except over the nucleus.

Acridine Orange

A weakly basic vital dye that concentrates in acidic compartments like lysosomes (emitting orange light upon blue/green excitation) and intercalates with RNA and DNA (emitting green light); it does not bind tightly to condensed chromatin.

How were the cells prepared in part two?

• We used dead cells because the stains that we used can’t cross the cell membrane

Direct Immunoflueorescence

• 1. PHEM Buffer (PIPES, HEPES, EGTA, MGSO4) 

  • Regulates pH

  • EGTA: chelates Ca2+ so that the cytoskeleton does not fall apart

  • MgSO4: to add Mg to keep cytoskeleton in polymeric form

• 2. Triton X-100

  • Non ionic detergent

  • destroys/dissolves membrane

• 3. Glutaraldehyde

  • Cross-links and freezes entire cytoskeleton

• 4. Blocking solution borohydride

• 5. Wash w/ PBS (phosphate buffer solution)

• 6A. Coomassie blue to stain entire skeleton

  • non-specific stain that binds generally to virtually all proteins present in a sample

• 6B. Alexa Fluor 488 Phalloidin to stain actin

  • Phalloidin: toxic b/c it binds to cytoskeleton

  • Alexa Fluor/Tas fluor: to dye cytoskeleton by covalently bonding to phalloidin

  • Blue excitation, green emission

Indirect Immunofluorescence Steps

1. Making the antibody: antigen injected into animal so that animal makes a primary antibody

2. Sample fixed so that antigen can bind to antibody

3. Primary antibody added to tissue

4. Secondary antibody w fluor added

   1. sec anti = raised in different species to target primary

   2. eg. goat anti-mouse antibody

5. Makes brighter signal and prevents harming primary’s ability to lock on target

Actin Filaments (Microfilaments)

appearing as super straight, sharp lines (or bundled stress fibers)

Function: help the cell move and change shape; aid in muscle contraction

• Stress Fibers: bundled actin cables that use energy to slide actin filaments past one another, allowing for contraction; also stabilize cell structure 

Microtubules (Tubulin)

more curvy but straight lines

Function: Creates a system of tracks within the cell allowing for things to be transported. They also position membrane-enclosed organelles inside the cell; help cells divide

Intermediate Filaments (Vimentin)

appearing as rigid or zig-zag lines

Function: Enables cells to withstand mechanical stress when cells are twisted or deformed; to prevent cell from tearing

Electron Microscopy (EM)

An imaging technique performed on preserved, non-living samples to examine ultrastructural details of cellular components at a $1\text{–}2\,\text{nm}$ resolution.

What cellular structure is this?

Plasma Membrane

What cellular structure is this?

Mitochondria

What cellular structure is this?

Smooth ER

What cellular structure is this?

Smooth ER

What cellular structure is this?

Rough ER

What cellular structure is this?

Golgi

Label

Label

What cellular structure is this?

Microtubules

What cellular structure is this?

Intermediate filaments (vimentin)