Cellular Processes: Protein Activity, Nuclear Transport, Endomembrane System, and Cytoskeleton

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Cell Processes and Lab Techniques

Differential Centrifugation

Differential centrifugation is a laboratory technique used to separate cellular components based on their density. This method is fundamental for isolating organelles and studying their functions in cell biology.

Principle: Centrifugal force separates particles by density; heavier components form a pellet, lighter remain in the supernatant.
Applications: Used to isolate nuclei, mitochondria, lysosomes, and other organelles for biochemical analysis.
Process: Sequential spins at increasing speeds allow stepwise separation of cellular fractions.

Example: Low-speed centrifugation pellets nuclei; higher speeds pellet mitochondria, lysosomes, etc.

Diagram showing centripetal and centrifugal forces in centrifugation Centrifuge with tubes Centrifuge schematic Stepwise separation of cell components by centrifugation

Protein Structure and Function

Protein Binding and Shape Change

Proteins are highly specific biological machines whose activity depends on their ability to bind substrates and undergo conformational changes. This specificity is crucial for cellular processes such as signaling, transport, and catalysis.

Binding Specificity: Proteins bind ligands, substrates, or signals via complementary chemical interactions (e.g., enzyme-substrate, receptor-signal).
Shape Change: Binding often induces a conformational change, altering protein activity.
Key Terms: Active site (region of enzyme where substrate binds), ligand (molecule that binds to a protein).

Example: Enzyme active site changes shape upon substrate binding, facilitating catalysis.

Enzyme-substrate binding Protein structure with active site Acetylcholinesterase active site binding Protein structure model

Nuclear Transport

Nuclear Pore Complex and Signals

The nuclear pore complex (NPC) regulates the entry and exit of molecules between the cytoplasm and nucleus. Proteins require specific signals to be transported across the nuclear envelope.

Nuclear Localizing Signal (NLS): A sequence of amino acids that directs proteins to the nucleus.
Nuclear Exit Signal (NES): A different sequence that facilitates export from the nucleus.
Shuttle Proteins: Bind to NLS and transport cargo through the NPC.

Example: Adding an NLS to a cytoplasmic protein causes it to localize in the nucleus.

Nuclear pore complex and transport Protein domain map with NLS NES sequence map Protein domain map with NLS and NES Protein sequence highlighting NLS Protein domain map with NES Shuttle protein transporting cargo through NPC Pyruvate kinase localization with NLS Fluorescent microscopy of protein localization

Endomembrane System and Protein Sorting

Protein Synthesis and Sorting

Proteins are synthesized on ribosomes and sorted to their correct cellular locations by signal sequences. The endomembrane system includes the ER, Golgi apparatus, lysosomes, and vesicles.

Cytosolic Ribosomes: Synthesize proteins for cytosol, mitochondria, chloroplasts, peroxisomes, and nucleus.
RER Ribosomes: Synthesize proteins for secretion, membrane, lysosomes, and ER.
ER Signal Sequence: Directs ribosome to the rough ER for co-translational import.

Example: Proteins with ER signal sequence are fed into the ER membrane during synthesis.

Protein synthesis and sorting diagram ER signal sequence schematic Signal hypothesis process Protein domain map with signal sequence

Pathway from ER to Outside of Cell

Proteins destined for secretion or membrane insertion follow a pathway from the ER to the Golgi apparatus, then to the plasma membrane or lysosome via vesicular transport.

Vesicular Transport: Proteins are packaged into vesicles that bud from the ER and fuse with the Golgi.
Sorting in Golgi: Proteins are modified and sorted based on amino acid tags.
Secretion: Vesicles fuse with the plasma membrane, releasing proteins outside the cell.

Secretory pathway step 1 Secretory pathway step 2 Secretory pathway step 3 Secretory pathway step 4 Secretory pathway step 5

Protein Sorting and Vesicle Formation

Specific amino acid tags direct proteins to their final destinations. Integral membrane proteins in the Golgi recognize these tags and facilitate vesicle formation.

Tag Recognition: Golgi membrane proteins bind to sorting sequences.
Vesicle Formation: Clusters of tagged proteins are packaged into vesicles by "pinching off" the membrane.

Protein sorting and vesicle transport Golgi sorting and vesicle formation Protein sorting schematic

Membrane Transport: Vesicles and Lysosomes

Vesicular Transport Mechanisms

Cells use vesicles to transport molecules across membranes. This includes endocytosis, exocytosis, pinocytosis, and phagocytosis.

Pinocytosis: Uptake of small dissolved substances or fluids.
Phagocytosis: Engulfment of large particles or entire cells.
Exocytosis: Release of molecules to extracellular space.
Endocytosis: Receptor-mediated uptake of specific molecules.

Vesicular transport schematic Pinocytosis schematic Phagocytosis schematic Receptor-mediated endocytosis

Pathways to the Lysosome

Lysosomes are cellular organelles responsible for degrading and recycling materials. There are three main pathways to the lysosome: receptor-mediated endocytosis, phagocytosis, and autophagy.

Receptor-Mediated Endocytosis: Molecules bind to cell surface receptors and are internalized.
Phagocytosis: Entire cells or large particles are engulfed and degraded.
Autophagy: Worn-out organelles are enclosed in vesicles and digested.

Receptor-mediated endocytosis pathway Phagocytosis pathway Phagocytosis schematic Autophagy pathway

Cytoskeleton: Structure and Function

Cytoskeletal Filaments

The cytoskeleton is a network of protein filaments that provides structural support, facilitates movement, and organizes cellular components. Most cells possess a cytoskeleton composed of actin filaments, intermediate filaments, and microtubules.

Actin Filaments: Thin, flexible fibers involved in cell shape, movement, and division.
Intermediate Filaments: Provide mechanical strength and anchor organelles.
Microtubules: Hollow tubes that support cell shape, transport vesicles, and form cilia/flagella.

Cytoskeleton in cells Cytoskeletal filament types

Filament	Subunits	Functions
Actin Filaments	Actin	Cell shape, movement, division
Intermediate Filaments	Keratin, lamins	Mechanical strength, organelle anchoring
Microtubules	α- and β-tubulin	Cell shape, vesicle transport, cilia/flagella

Motor Proteins and Cellular Movement

Motor proteins such as myosin, kinesin, and dynein interact with cytoskeletal filaments to generate movement within cells.

Myosin: Pulls on actin filaments for muscle contraction and cell movement.
Kinesin and Dynein: Move along microtubules, transporting vesicles and organelles.
Cilia and Flagella: Motor proteins drive movement of these structures for cell motility.

Myosin motor protein pulling on actin

Summary Table: Cytoskeletal Filaments

Type	Diameter	Subunit	Function
Actin Filaments	7 nm	Actin	Cell shape, movement, division
Intermediate Filaments	10 nm	Keratin, lamins	Mechanical strength, anchoring
Microtubules	25 nm	α- and β-tubulin	Cell shape, transport, cilia/flagella

Cytoskeletal filament types

Additional info: Cytoskeletal filaments are dynamic structures, constantly assembling and disassembling to facilitate cellular processes such as mitosis, intracellular transport, and cell migration.