Cellular Transport: Microfilaments

• Cellular Transport and Cytoskeleton Part 1 includes microfilaments, intermediate filaments, and microtubules.

Microfilaments

• The major component of microfilaments is actin.
• G-actin is the globular monomer of microfilaments.
• F-actin is the filamentous polymer, formed from the polymerization of G-actin.
• ATP hydrolysis is needed for G-actin monomers to bind to the growing chain.

Assembly of Actin Filaments

• A trimer forms from three single G-actin monomers during the process.
• F-actin grows through barbed or plus ends, growing faster, or pointed or minus ends, growing slower.
• Filaments are polar because the two ends grow at different rates.

Organization of Actin Filaments

• Microfilaments can be organized into bundles or networks.
• Small-rigid proteins act as cross-linking proteins to align filaments.
• Fimbrin is an example that forces close alignment in intestinal microvilli.
• Alpha-actinin is present when spacing is wider, allowing myosin to interact with actin filaments during muscle contraction.
• Large-flexible proteins can form networks; filamin can be found in macrophages.

Functions of Microfilaments

• Maintains the cellular structures in the plasma membrane, like the erect position of microvillus.
• Facilitates muscle contraction, especially with cross-bridge formation of actin and myosin filaments.

Intermediate Filaments

• All are fibrous, not globular, allowing them to form a fiber-like structure with a 12 nm diameter compared to microfilaments which are 7 nm and microtubules which are 25 nm.
• Tetramer assembly forms protofilaments, which build a filamentous structure.
• Intermediate filaments can be cytoplasmic, like keratin and vimentin, or nuclear, like neurofilaments and nuclear lamin.

Assembly of Intermediate Filaments

• Polypeptides link to form a dimer, which will coil.
• The dimers link to form tetramers.
• Protofilaments then form from the tetramers.
• Eight protofilaments wind together to form an intermediate filament.

Intermediate Filaments in Specific Tissues

• Provide mechanical strength.
• Epithelial cells contain keratin.
• Connective tissues contain vimentin.
• Neural fibers contain neurofilaments.
• Nuclear pores contain nuclear lamins.

Microtubules

• Composed of alpha- and beta-tubulin heterodimers with a large 25 nm diameter compared to microfilaments which are 7 nm and intermediate filaments which are 12 nm.
• Located at the centrosome in animal cells and associated with the nuclear membrane during prophase stage of mitosis.

Microtubules plus/minus ends

• Tubulin dimer containing GTP is added to the microtubule during polymerization.
• GTP on beta-tubulin is hydrolyzed into GDP and a phosphate group after binding to the growing chain.
• Protofilaments with GDP are unstable and can peel away from the microtubule wall.
• Beta-tubulin is exposed at the plus end while alpha-tubulin, which forms heterodimers with beta-tubulin, is exposed at the minus end.
• Movement exists from (+) to (-) or reverse to maintain polarity and separation of chromosomes during cell division.

Dynamic Instability of Microtubules

• GTP numbers will influence directional results of elongation vs shortening.
• Microtubule associated proteins are present at the dimer to stabilize and facilitate polymerization.
• Dimer prevents the fall of the plus end like a "helmet".
• Without the cap, dimer, or a short chain of subunits will dissociate.

Functions of Microtubules

• Protein and lipids are synthesized in the cell body and transported to the synaptic terminal with the help of motor proteins.
• Kinesins move towards the plus (+) end of organelles.
• Dyneins move towards the minus (-) end of organelles.
• There are also dynein and kinesin members which can either move towards the plus or minus end.
• During mitosis, separates chromosomes: microtubule forms a mitotic spindle to guide chromosome movement and segregation.
• Cell locomotion: flagella associate motor proteins of sperm for motion.

Agents that Prevent Microtubular Functions

• Colchicine prevents dimer assembly into microtubules.
• Vincristine and vinblastine prevent polymerization of tubulin.
• Taxol prevents the depolymerization of tubulin.

Overview of Cytoskeletal Filaments

• Microfilaments use ATP, are yes for polarity, and use myosin for motor proteins.
• Microtubules use GTP, are yes for polarity, and use kinesin/dynein for motor proteins.
• Intermediate filaments use none for nucleotide, motor proteins, and no for polarity.
• Microfilaments and microtubules are considered highly dynamic, while intermediate filaments are less dynamic.
• Major functions include muscle contraction/cell shape for microfilaments, cell and tissue integrity for intermediate filaments, and organization/long-range transport for microtubules.
• Cellular Transport and Cytoskeleton Part 2 includes secretory or exocytosis, endocytosis, protein sorting, and secretion.

Overview of Secretory & Endocytic Pathways

• Includes exocytosis when a secretory vesicle binds to the membrane and releases an extracellular matrix protein.
• Includes endocytosis using constitutive and regulated secretory pathways for example, releasing insulin from pancreatic beta cells.

Secretory Pathways

• In the process, protein inserts itself into the endoplasmic reticulum membrane before forming vesicles to exit the ER.
• Proteins move outward to the cell surface from the cis- to trans-face of the Golgi apparatus within the vesicle.
• Proteins either continuously secrete, or hormonally stimulate a pathway.

Endocytic Pathway

• Macromolecules will bind to the cell surface which can create a clathrin-coated vesicle, called an endosome.
• Macromolecules separate from the receptor but are still in the cell membrane for it to be recycled later.
• The endosomal membrane will fuse with the lyosomal to transport the macromolecule.

Rough Endoplasmic Reticulum

• Ribosomes make the structure rough to assist protein synthesis.
• Functions for production and modification occur for proteins like plasma membrane, lysosomal, and secretory proteins.
• Transports and releases vesicles to the Golgi apparatus for further modification.

Translation

• mRNA production occurs in the nucleus which then moves through the nuclear pore into the cytoplasm.
• Ribosomes join in the cytoplasm to activate protein synthesis.
• The receptor, which attaches in the rER membrane, transports the protein into the ER lumen.

Golgi Apparatus

• Major functions include processing and protein sorting.

Protein Sorting - Pathway A vs B

• In Pathway A, ribosomes synthesizing nascent proteins are directed to the rough ER by the presence of an ER signal sequence.
• These proteins will then move into the Golgi complex, which will sort everything.
• In Pathway B, proteins are released into the cytosol after freeing themselves from the ER signal sequence.
• They are imported into the mitochondrion, nucleus, and peroxisome respectively via organelles.

Secretory Proteins Released from the Cell by Exocytosis (Pathway A 4c)

• Includes the release of external proteins from the beta cells contained pancreatic fluid.
• Insulin is only released during high levels of glucose.

Endocytosis

• Includes phagocytosis, pinocytosis and receptor-mediated endocytosis.
• Phagocytosis is specific to phagocytes for solid ingestion.
• Pinocytosis is a eukaryotic cell involving the ingestion of liquid.

Endocytosis: Receptor-Mediated Endocytosis

• Receptors, once bound with macromolecules, form a coated vesicle with aid of Adaptor Proteins
• Clathrins will break down and recycle adaptors.
• Uncoated then fuses the macromolecule to its final designation.

Clathrin-Coated Vesicles

• Complexes connected to Clathrin triskelions, a three legged structure, will automatically internalize.

Receptor-mediated Endocytosis in Low Density Lipoprotein (LDL) Pathway

• With LDL, protein binds and the ligand becomes an enclosed vesicle.
• The clathrin then depolymerizes to become an early endosome.
• Low pH later prompts LDL particles to break down back to cholesterol and amino acids.

Summary

• Secretory pathway and regulated proteins are "going out" whereas, endocytic pathways are "getting in".
• Ribosomes and Golgi bodies have transmembrane faces, which synthesize protein directing proteins to the rough ER with aid of ER signal sequences.
• All of this is occurring with the help of organelle: Endocytosis and Lysosomes.