Cell stuff: 9

Cytoplasm and Cytosol

• Cytosol is the fluid contained within the cell membrane.
• Cytoplasm comprises the entire content within the cell membrane.

Light Microscopy

• Light microscopy relies on the contrast created by the combination of waves out of phase.
• Wavelength of light limits light microscopy resolution, making it difficult to observe objects smaller than the wavelength of light.

Fluorescence Microscopy

• Fluorescence microscopy uses photons, which have energy and wavelength dependent on their energy level.
• In fluorescence microscopy, fluorescent molecules absorb a photon with specific energy, causing an electron to excite to a higher energy orbital.
• The excited electron then falls back to the ground state, releasing energy as light with a longer wavelength.

GFP (Green Fluorescent Protein)

• GFP allows scientists to make proteins fluorescent.
• GFP gene is localized within the cell and fused with the gene for a targeted protein.
• The cell then synthesizes the target protein with fluorescent properties.

Electron Microscopy

• Electron microscopy uses electron bundles instead of light.
• Electron microscopy provides a much higher resolution (around 1nm) compared to light microscopy, allowing the observation of smaller structures.
• Electron microscopy requires specialized techniques like ultra-thin sample cutting and is generally more expensive than light microscopy.

Membranes

• Membranes are formed by lipids in water.
• Lipid bilayers typically measure 5 nm in thickness.
• Biological membranes consist primarily of lipids for structural support and proteins.
• Membranes are a bilayer, with lipids arranged in two opposing layers.

Membrane Importance

• Membranes are crucial for defining cell boundaries and compartmentalizing organelles.
• Lipids can diffuse fluidly across the membrane.

Membrane Transport

• Passive diffusion allows molecules like oxygen and hydrophobic molecules to pass through the membrane.
• Hindered diffusion facilitates the movement of small uncharged polar molecules like water.
• Transporter diffusion is specifically used for ions like magnesium and calcium.

Membrane Proteins

• Membrane proteins function as transporters and channels.
• They facilitate communication between the cell's interior and exterior.
• They help localize proteins at the cell surface and in the cytosol.

Beta-barrels

• Beta-barrels are a structural motif in membrane proteins.
• They have hydrophobic amino acid side chains on the outside and polar side chains on the inside.

Lipid Bilayer Composition

• The hydrophilic group of phospholipids contains choline, phosphate, and glycerol.
• The hydrophobic tail can vary depending on the presence of double bonds, impacting lipid interactions and membrane structure.
• Cholesterol increases membrane stability and decreases permeability.

Membrane Asymmetry

• Phospholipids on the cytosol side of the membrane are more negatively charged.
• Phospholipids on the extracellular side are more neutral or positively charged.
• Sugars attach to the polar head groups of phospholipids on the extracellular side, providing protection and facilitating cell recognition.

Polarized Cells

• Polarized cells have distinct regions with specific functions.
• The apical plasma membrane is responsible for protein and vesicle secretion.
• The lateral plasma membrane connects with other cells.
• The basal plasma membrane is firmly attached to the basal lamina.

Cytoplasm and Cytosol

• Cytosol is the fluid contained within the cell cytoplasm, surrounding cell organelles in eukaryotes.
• Cytoplasm is the entire content within the cell membrane.

Light Microscopy

• Waves out of phase generate contrast when combined.
• A wave's phase shifts due to the density of the specimen it passes through.
• Light microscopy is limited by the wavelength of photons, unable to observe objects smaller than the wavelength of light shone upon it.

Fluorescence Microscopy

• A photon (light particle) possesses energy, with higher energy photons having shorter wavelengths.
• When a photon with the correct energy hits a fluorescent molecule, an electron is excited to a higher energy orbital.
• This excited electron rapidly transitions to a slightly lower energy orbital.
• Finally, the electron falls back to the ground state, emitting residual energy as light.
• The emitted light has lower energy and therefore a longer wavelength.

Fluorescent Microscopy Components

• First barrier: Blocks unnecessary wavelengths, allowing blue light to pass between 450-490 nm.
• Dichroic mirror (beam-splitting mirror): Reflects light below 510 nm and transmits light above 510 nm.
• Objective lens: Ensures visibility of the object.
• Second barrier: Cuts out unnecessary fluorescent signals.
• GFP: Green fluorescent protein.

Making a Protein Fluorescent

• Localize the GFP gene.
• Fuse the gene for the protein with the GFP gene, forming one larger gene.
• The cell will synthesize the protein with fluorescent behavior.

Electron Microscopy

• Uses electron bundles instead of a light source.
• Resolution is approximately 1 nm, compared to about 200 nm for light microscopy, allowing observation of much smaller objects.
• EM is significantly more expensive than light microscopy.
• EM requires ultra-thin cutting of samples.

Membranes

• Lipids in water form membranes.
• The size of a lipid bilayer is typically 5 nm.
• Biological membranes surround cells or cell organelles.
• Biological membranes consist primarily of lipids for structure and proteins.
• Biological membranes are a bilayer.

Membrane Importance

• Essential for forming boundaries between cells and compartmentalizing organelles.
• Lipids fluidly spread across the membrane.

Membrane Diffusion Types

• Passive diffusion: Oxygen and other hydrophobic molecules.
• Hindered diffusion: Small uncharged polar molecules, such as water.
• Transporter diffusion: Ions (e.g., magnesium or calcium).

Membrane Proteins

• Include transporters and channels.
• Facilitate communication between the outside and the intracellular environment.
• Localize proteins at the cell surface.
• Localize proteins at the membrane in the cytosol.

Beta-Barrel Structure

• Amino acid side chains are hydrophobic on the outside and polar on the inside.

Hydrophilic Groups

• Choline: Differs between negative and positively charged environments.
• Phosphate:
• Glycerol:

Hydrophobic Tail

• The tail can affect the structure of multiple lipids when there is a double bond, altering the formation of the hydrophobic tail and leading to looser bonds between lipids.

Cholesterol

• Increases the stability of the membrane.
• Decreases membrane permeability.

Bilayer Phospholipids

• Phospholipids at the cytosol side are more negatively charged.
• Phospholipids at the extracellular side are more neutral or positively charged.
• Sugars attach to the polar head groups at the extracellular side, protecting the membrane from the outside and facilitating cell recognition.

Polarized Cell

• Secretes proteins and vesicles at the apical plasma membrane.
• Connects with other cells at the lateral plasma membrane.
• Is firmly bound to the basal lamina at the basal plasma membrane.

Voltage-Gated Sodium Channels

• Proteins that open in response to changes in membrane potential, allowing sodium ions to flow into the cell.
• Enable propagation of action potential in only one direction.

Voltage-Gated Sodium Channel Mechanism

• Opens in response to membrane depolarization, allowing Na+ ions to flow into the cell.
• Enters a refractory state where it is inactivated.
• Returns to a closed form to receive another signal.

Repolarization

• After sodium rushes in, potassium rushes out, causing repolarization.

Sodium-Potassium Concentration Restoration

• Achieved using the sodium-potassium pump.
• Exchange of 3 Na+ for 2 K- requires one ATP, and glucose can be used as a tracer.

This quiz covers key concepts about cytoplasm, cytosol, and various microscopy techniques including light and fluorescence microscopy. It also explores the use of GFP in cellular studies, emphasizing its importance in modern biology. Test your understanding of these critical topics in cell biology!