Cellular Form and Function in Eukaryotic Cells

Section 2 Overview

• Section 2 will cover cellular form and function in eukaryotic cells.
• Emphasis is placed on identifying cell parts, matching processes to cell parts, contextualizing processes, and recognizing experimental techniques.

Cell Architecture

• Eukaryotic cells contain DNA, mRNA, and proteins.
• Cytoskeleton roles are covered in Week 4.
• Organelles & Endomembrane system functions explained week 3
• Junctions, adhesion, and ECM are covered in Week 5
• Membrane structure and transport are covered in Weeks 1 and 2.
• Cell Cycle is covered in Week 6

Saccharomyces cerevisiae

• Saccharomyces cerevisiae (yeast) is a model eukaryote.
• Its cell structure includes a cell wall, Golgi stack, nucleus, mitochondrion, ribosomes, and a plasma membrane.

Multicellular Organisms Cell Types

• Mammalian skin contains various cell types, including epidermis, dermis, and hypodermis tissues.
• Skin cells include keratinocytes, melanocytes, fibroblasts, and immune cells like Langerhans cells.
• Plant cells include mesophyll cells, guard cells, trichomes and root hair cells

Animal Cell Architecture

• Animal cell architecture includes the extracellular matrix and lysosomes
• Extracellular matrix contains specialized material outside the cell
• The lysosome is responsible for the Degradation of cellular components that are no longer needed

Plant Cell Architecture

• Plant cells feature a cell wall for cell shape and mechanical stress protection.
• Plant cells contain two types of Vacuoles, one for degradation, and one for the storage of molecules and proteins
• Chloroplasts are sites of photosynthesis

Eukaryotic Cell Components

• Cytoplasm consists of the contents of the cell outside the nucleus.
• Cytosol is the aqueous part of the cytoplasm.
• Lumen exists inside of organelles

Membrane Functions

• Membranes act as selectively permeable barriers.
• Membranes help in the the transport of solutes.
• Membranes are responsible for interactions between cells and compartmentalization.
• Membranes provide a scaffold for biochemical activities, and for responding to external signals.

Membrane Bilayers

• Singer and Nicolson proposed the Fluid Mosaic Model of the Membrane in 1972.
• Cell membranes consist of a lipid bilayer in which proteins are embedded.
• The lipid bilayer has 5nm thickness
• The membrane contains two layers or leaflets
• Membrane molecules are amphipathic, having hydrophilic (polar) head groups and hydrophobic tails.

Membrane Lipids Composition

• Membranes are assembled from many different lipids: Phospholipids, Sterols, and Glycolipids
• The common phospholipid phosphatidylcholine has a polar head (choline), phosphate and glycerol group
• The tail is unsaturated and contains a has a cis-double bond

Membrane formation

• In aqueous environments, phospholipids spontaneously self-associate into a bilayer.
• Polar head groups interact with water.
• Hydrophobic hydrocarbon tails interact with each other.

Liposomes

• Liposomes are artificial lipid bilayers.
• Liposomes have 3 key uses: to help study lipid properties, help with membrane protein properties and to help deliver drugs into cells

Cell Membrane Fluidity

• Cell membranes can be deformed without causing damage.
• Laser tweezers can manipulate the membrane
• Within each leaflet, phospholipids can diffuse laterally, rotate, and flex rapidly.
• Phospholipids rarely move from one leaflet to another (flip-flop).
• Membrane fluidity is important for function and is carefully regulated
• This allows movement of membrane proteins for transport, enzyme activity, and signaling.

Membrane Fluidity Factors

• Membrane fluidity is affected by temperature and the composition of the phospholipid saturation and tail length
• Lower temperatures make the membrane more viscous.
• Cis-double bonds increase fluidity at lower temperatures (reducing tight packing).
• Shorter hydrocarbon tails increase fluidity at lower temperatures (lipid tails interact less).
• Cholesterol in animal cell membranes stiffens the membrane, making it less permeable to water.

Sterols

• Cholesterol is the main sterol in animal cell membranes.
• Plants have plant sterols in some cholesterol
• Cholesterol content can reach up to a 1:1 ratio with phospholipids.
• Cholesterol decreases mobility of phospholipid tails and reduces the plasma membrane's permeability to polar molecules.

Lipid Movement

• Scramblase is a phospholipid translocator that catalyes rapid flip-flops of random phospholipids from one leaflet to the other in the ER membrane
• This occurs in the cytosolic leaflet of the endoplasmic reticulum

Lipid Bilayer Asymmetry

• Membranes retain their orientation
• The membrane has a noncytosolic face/leaflet (red) and cytosolic face/leaflet (pink)
• Flippases catalyzes flip-flops of specific phospholipids to the cytosolic leaflet
• Glycolipids and glycoproteins are asymmetric in bilayers
• There is the presence of phosphatidylserine which causes asymmetry

Glycolipids and Glycoproteins

• Glycolipids and glycoproteins protect the membrane from harsh environments and end up at plasma membrane inside of some organelles
• Glycolipids and glycoproteins noncytosolic face

Membrane Protein Overview

• Membrane proteins have specific orientations that are essential for function
• Membrane proteins can be associated with the lipid bilayer in different ways.
• membrane proteins have to either be inserted into the bilayer or attached to a lipid which is inserted into the lipid bilayer.

Intergral vs Peripheral Proteins

• Integral membrane proteins are inserted into the lipid bilayer or attached to lipids.
• Extraction methods for integral proteins use detergents, which destroy the lipid bilayer
• Peripheral proteins don't insert into the membrane, they are on either face of the membrane
• Peripheral protein are bound to other proteins or specific lipids
• Peripheral proteins use gentle extraction methods for extraction (lipid bilayer is intact)

Transmembrane Protein Traits

• Proteins can be either a single a-helix, multiple a-helices, or a rolled B-sheet
• Transmembrane proteins are amphipathic.
• They have hydrophilic (polar) domains and hydrophobic (non-polar) membrane-spanning domains.

Membrane-Spanning Domain Examples

• Membrane-spanning alpha-helix domains are are about 20 hydrophobic amino acids
• Single alpha helices span across the membrane
• Multiple alpha helices for a hydrophilic pore to pass through the membrane
• B-barrel creates a rigid channel

Functions of membrane proteins

• Transporters and Channels
• Anchors
• Receptors
• Enzymes

Structure Identification Techniques

• X-ray crystallography determines 3D structure of the membrane
• Hydrophobicity plots segments of 20-30 hydrophobic amino acids can span the lipid bilayer as an α-helix.

Monolayer-Associated Membrane Proteins

• Proteins anchored on the cytosolic face by an amphipathic alpha-helix
• Proteins in membrane binding (Sar1) helps vesicle budding at the ER

Lipid-Linked Membrane Proteins

• GPI anchors have synthesis in ER lumen that end up on the cell surface
• Lipid anchor that have cytosolic enzymes adds a protein that directs towards the the cytosolic face.

Technique: Extraction

• Mild detergents can make proteins solubilize in water

Studying Membrane Protein Properties

• Mild detergents can be used to solubilize and reconstitute functional membrane proteins.

Lateral Diffusion of Membrane Proteins

• Lateral diffusion within the leaflet
• Study of protein movement by: Fluorescence Recovery After Photobleaching (FRAP) uses a Green Fluorescent Protein

FRAP

• Proteins fused to GFP or labeled with fluorescent
• A laser can create a bleach patch
• Protein will diffuse randomly from outside the patch and cause Fluorescence to comeback to the bleached patch
• The time taken for migration measures proteins is the taken for the neighboring unbleached fluorescent proteins to move into bleached area

2008 Nobel Prize in Chemistry

• The 2008 Nobel Prize in Chemistry recognized the discovery and development of the green fluorescent protein, GFP.