Cellular and Molecular Biology Topics
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Prokaryotic vs Eukaryotic Cells
Prokaryotic cells are much smaller than eukaryotic cells (1-2 mm vs. 5-100 mm) and can have either anaerobic or aerobic metabolism, while eukaryotic cells are almost always aerobic.
The DNA of prokaryotes is usually haploid and circular, without chromatin. Eukaryotes have diploid DNA (except germ cells) with introns, organized into chromosomes and chromatin. Prokaryotes also lack the extensive cytoskeleton substructure present in eukaryotes.
Prokaryotic cells have an outer plasma membrane, a cell wall and an inner plasma membrane surrounding the cytoplasm, were all chemical reactions occur. Eukaryotic cells have a single plasma membrane and internal membrane-bound organelles which segregate specialized subcellular functions.
The largest organelle of the eukaryotic cell is the nucleus, which contains the genetic information of the cell and is the site of DNA replication and RNA synthesis. The translation of RNA into proteins takes place in the cytoplasm. Extending from the nuclear membrane is the endoplasmic reticulum, an extensive network of intracellular membranes. From the ER, proteins are transported in small vesicles to the Golgi apparatus, were they are further processed and sorted for transport to their final destinations. The Golgi is also the site of lipid synthesis. The mitochondria, is the site of oxidative metabolism and generates most of the ATP. Lysosomes and peroxisiomes provide specialized metabolic compartments for the digestion of macromolecules and for various oxidative reactions, respectively. The cytoskeleton provides the structural framework of the cell, and is a network of protein filaments extending through the cytoplasm.
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The Nucleus
The
nucleus is surrounded by the nuclear envelope, which is a double membrane structure
continuous with the ER. The outer nuclear membrane has docking proteins mediating
ribosome binding to the outer leaflet. The inner nuclear membrane is supported
by lamins, nuclear intermediate filament proteins that polymerize in a 2-D lattice
that gives shape and stability to the nuclear envelope.
Inside the nuclear laminae is the chromatin: a complex of DNA, histones and non-histone proteins which form the eukaryotic chromosome. The euchromatin is the less condensed portion of the chromatin, including most active genes. The heterochromatin are the regions of highly condensed chromatin which are transcriptionaly inactive.
The nucleolus is the site of rRNA synthesis and processing, where the ribosomal units are assemble. It consists of large loops of DNA emanating from several chromosomes, each of which contains a cluster of rRNA genes. Each such gene cluster is known as a nucleolar organizer region. In an electron micrograph, three partially segregated regions can be distinguished in the nuclear organizer region: the fibrillar center contains DNA that is not being transcribed, the dense fibrillar component contains RNA in the process of being synthetized, and the granular component contains the maturing ribosomal precursor particles.
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The Tissue Matrix
The tissue matrix is a dynamic linkage between structural proteins of the extracellular matrix, the cytoplasm and the nucleus. The nuclear matrix is a proteinaceous substructure of the nucleus that resists nuclease digestion and high salt extraction.
Over 6 feet of DNA is organized in the 10 µm diameter of the nucleus by supercoiling wrapping around nucleosomes, which are made of histone proteins. The nucleosomes get arranged into filaments, like beads on a string, which are then arranged into loops to form chromosomes. The most tightly package strands of DNA are less available for transcription, while loose loops are the active genes. How loose or tight a strand of DNA is coiled is determined by the Matrix Attachment Region (MAR) protein, which also attach the chromosome to the nuclear matrix.
MAR proteins topoisomerase II alpha and beta regulate DNA supercoiling. There are also Nuclear Matrix proteins (NuMa) that form the mitotic spindle pole apparatus in dividing cells. The inner nuclear envelope is supported by lamins, nuclear intermediary filament proteins which polymerize in a 2-D lattice, giving shape and stability to the nuclear envelope.
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Nuclear Pore Complex
The nuclear pore complex
(NPC) is composed of multiple copies of about 50-100 proteins called nucleoporins.
These 
proteins
form several structures of the NPC:
Ions, metabolites and small proteins (<60 KD) diffuse freely through nuclear pores, while macromolecules larger than 60 KD must be actively transported by a process requiring ATP. Proteins to be actively transported inside the nucleus have a nuclear localization signal (NLS). Proteins to be actively exported from the nucleus contain a nuclear export signal (NES). Shuttling proteins often contain both NLS and NES.
The NES mechanism occurs as follows:
The NLS mechanism occurs as follows:
In general, GTP-binding proteins act as molecular on/off switches in signal transduction patways. Proteins are turned “on” when bound to GTP and turned “off” when bound to GTP. A signal activates the release of GDP and subsequent binding of GTP. The switch proteins promote the activity of specific effector proteins by direct protein-protein interactions. The binding region in in its active conformation only when the switch protyein is bound to GTP. Ran is an example of a GTPase switch protein.
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Membranes
Membranes are composed of a lipid bilayer: two leaflets of mostly phospholipids
arranged according to hydrophobic interactions.
The fatty acyl tails of phospholipids are arranged into a hydrophobic core while
the polar heads of one leaflet are in the cytoplasm and the heads of the other
leaflet are in the extracellular fluid.
While the major driving force forming the bilayer are the hydrophobic interactions between fatty acid chains, van der Waals interactions among hydrocarbon chains favor closer packing of the hydrophobic tails.
Lipid membranes also contain glycolipids, cholesterol and proteins. The chemical composition of membranes varies from cell to cell. Most have more lipids than proteins and more proteins than carbohydrates. The mitochondrial inner membrane has more proteins than lipids and no carbohydrates.
Each leaflet of the lipid bilayer often exhibits a distinct lipid and glycolipids composition. Lipids with positively charged head groups (phosphatidylcholine) or sphingomyelin are observed in the exoplasmic leaflet, while neutral or negatively charged lipids (phosphatidylserine, phosphatidylethanolamine) are found in the cytoplasmic leaflet. Sugar residues occupy the exoplasmic leaflet or the non-cytoplasmic luminal side of organelles.
Thermal motion allows membrane lipids to rotate freely around their long axis and to diffuse laterally within a leaflet. A lipid can diffuse the length of the eukaryotic cell within 20 seconds.
Phase transition is due to increased motion about the C-C bonds of fatty acyl chains, which pass from a highly ordered, gel-like state to a more fluid state. This transition occurs over a narrow temperature range, around the melting point. Short and unsaturated fatty acyl chains undergo phase transition at lower temperatures than long, saturated chains.
Maintenance of the bilayer fluidity is essential for cell growth and division. In a lipid bilayer, the rigid hydrocarbon rings of cholesterol interact with the fatty acid chains of adjacent phospholipids near their polar heads, decreasing the mobility of the outer portions of the fatty acid chains, making this part of the membrane more rigid but still maintaining fluidity at lower temperatures. Cholesterol is a major determinant of fluidity, since it separates and disperses tails of fatty acids. At 37°C cholesterol restrict fluidity. Lipids containing unsaturated fatty acids also increase membrane fluidity because the double bonds create kinks in the fatty acid chains, making them more difficult to pack together. Take Quiz: [Q1] [Q2] [Q3]
Fluid Mosaic Model
The
current model of membrane structure views membranes as a fluid mosaic in which
proteins are inserted into a lipid bilayer. While phospholipids provide the
basic structural organization, proteins inserted in the lipid bilayer carry
out specific functions of membranes in different cells.
An integral protein is a membrane-bound protein, all or part of which interacts with the hydrophobic core of the phospholipids bilayer and can be removed from the membrane only by extraction with detergent. Peripheral or extrinsic proteins associate with either the cytosolic or exoplasmic face of a membrane but do not enter the hydrophobic core of the membrane, and may be easily removed from the membrane. Protein kinase C is an example of a peripheral protein.
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Integral proteins have hydrophilic regions which protrude into either the aqueous extracellular fluid or the cytoplasm, and hydrophobic regions that interact with the fatty acyl chains of the lipid bilayer. Integral proteins that stretch across the entire bilayer and project on both sides of the membrane are known as transmembrane proteins. Often, the hydrophobic portion of the protein consist of an alpha-helix.
Some integral protein’s amino acid chain may loop back and forth across the membrane several times, alternating hydrophobic alpha-helix segments with polar portions that protrude into the aqueous fluid on either side. Others are anchored to one of the membrane leaflets by a covalent bond to a lipid chain. The polypeptide chain does not enter the lipid bilayer. For example, Ras is covalently linked to the cytosolic face by a 15-carbon farnesyl moiety. Alkaline phosphatase is covalently linked to the exoplasmic face by a fatty acid.
Glycoproteins have a carbohydrate groups attached to the ends that protrude into the extracellular fluid. They play a role as molecular “signatures” that enables cells to recognize one another. They also enable cells to adhere to one another and protects the cell from being digested by enzymes in the extracellular fluid.
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Endoplasmic Reticulum and Golgi Apparatus
Typically the largest membrane in eukaryotic cells, the endoplasmic reticulum is a compartment comprising a network of interconnected, membrane-bound vesicles known as cisternae. The rough endoplasmic reticulum is contiguous with the nuclear envelope and is studded with ribosomes. The smooth endoplasmic reticulum lacks ribosomes and is commonly tubular in form.
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The rough ER is the site of synthesis of plasma membrane and organelle proteins, nearly all secreted proteins, and proteins of the extracellular matrix. Major functions include glycosylation, disulfide bond formation, polypeptide folding and protein subunit assembly.
The smooth ER is the main site of fatty acids and phospholipids synthesis, therefore it is prominent in cells involved in lipid metabolism and steroid secretion. Enzymes in the smooth ER of hepatocytes detoxify hydrophobic chemicals. The smooth ER is also prominent in striated muscle.
The Golgi apparatus is a series of flattened membrane vesicles or sacs, surrounded by a number of spherical membrane vesicles. In secretory cells, for example hepatocytes, the Golgi has many flattened layers known as cisternae or saccules. Nonsecretory cells, for example fibroblasts, have few saccules.
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The Golgi apparatus has three defined regions: cis Golgi, medial Golgi, and trans Golgi. Each region has distinct enzymes that modify proteins differently depending on their structure and final destination.
The cis Golgi resides closest to the ER. Transfer vesicles from the rough ER fuse with the cis Golgi and deposit proteins. There are a variety of functional transfer vesicles, coated with specific protein complexes, associated with the Golgi and ER. For example clathrin targets cargo from Golgi to plasma membrane, COPI from ER to ER, and COPII from ER to Golgi.
The median Golgi is the middle portion of the stacks, responsible for the glycosylation of proteins and lipids and the assembly of proteoglycans. For example, the addition of mannose 6-phosphate is a targeting signal for enzymes destined to the lysosome.
The trans Golgi, the most distal to the ER, Golgi sorts and packages proteins for transport to their destination organelles.
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Endocytosis
The uptake of materials by cells from the extracellular or intracellular space involves the isolation and degradation of materials by three different routes: endocytosis, autophagy and lysosomal targeting.
Endocytosis is the uptake of extracellular materials by invagination of the plasma membrane to form a small membrane-bound vesicle. Endocytosis can occur as pinocytosis, receptor-mediated endocytosis and phagocytosis.
In receptor-mediated endocytosis, a specific receptor on the cell surface bind tightly to an extracellular macromolecule that it recognizes as ligand. The plasma membrane region containing the receptor-ligand complex then undergoes endocytosis facilitated by clathrin-coated pits. The pits form from the cytosolic surface of the plasma membrane or the trans Golgi.
Purified clathrin forms a triskelion: a three-limb structure, each limb with one light and one heavy chain.
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The triskelions polymerize to form a cage-like structure around the membrane. Assembly particles contain one copy each of a different adapter protein that binds to the globular domain of each heavy chain and promote clathrin polymerization.
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The adapter proteins also bind to specific cytosolic-face membrane proteins, determining the region of membrane to bud. Dynamin, a protein with GTPase activity, requires GTP to mediate the pinching of the vesicle.
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Pinocytosis, “cell drinking”, is a clathrin-independent endocytosis, involving non-specific uptake of small droplets of extracellular fluid into endocytic vesicles. Phagocytosis, “cell eating”, is the engulfing of large particles such as bacteria by a section of the plasma membrane. Autophagy is the engulfment and digestion of a defective cellular organelle by another.
Lysosomal targeting occurs when a cytosolic protein carrying a targeting signal (Lys-Phe-Glu-Arg-Gln) becomes aged, and is dragged into a lysosome for destruction.
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Lysosomes
After a clathrin coated endocytosis vesicle is separated from the membrane, the clathrin depolymerizes. The membrane bound vesicle discharges its content into the early endosomes. The early endosomes is a tubular vesicle just below the plasma membrane, with its own set of proteins and pH about 6.2 to 6.5 .
The early endosomes is the main sorting station of the cell. The membrane receptor will be recycled back to the plasma membrane, while the engulfed particle may have one of two fates: can be transcytosed or degraded. Transcytosis is the transfer of the engulfed particle to the opposite domain of the plasma membrane.
Lysosomes are the major digestive organelle of the cell (0.2 – 0.5 mm in diameter). They are vesicles bound by a single membrane and contain a large number of hydrolases. They are involved in receptor-ligand degradation, cholesterol metabolism (via the LDL receptor) and organelle turnover. The lumen pH is low, about 5, maintained by an ATP-driven proton pump.
Familial hypercholesterolemia is a disease characterized by high levels of cholesterol in blood. Homozygous for the mutant allele die at an early age from heart attacks. In one form of the disease, the LDL receptor cannot be internalized into clathrin-coated pits. The cytosolic domain of the LDL receptor is missing the required amino acid sequence fro linking to the clathrin coat: Tyr-X-X-$ ($ = any bulky hydrophobic amino acid). In I cell disease, hydrolases destined for the lysosome are not properly tagged and do not make it to the organelle. Inclusion bodies are prevalent in people with this disease, which leads to neurological disorders, mainly mental retardation, and early death.
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Peroxisomes
Peroxisomes are single membrane-bound organelles, 0.2-1 mm in diameter. They appear singular in cross section but serial sectioning reveal a reticular form. Some peroxisomes have a crystalline core largely composed of urate oxidase, an oxidative enzyme. Usually peroxisiomes in different cells of the same organism contain different enzymes.
Peroxisomes oxidize organic substrates, particularly the breakdown of fatty acids into 2-carbon fragments (beta-oxidation) that are converted to coenzyme A and exported from the peroxisome. Other oxidative reactions mediate the detoxification of various molecules in the liver and kidneys.
Molecular oxygen is used to remove hydrogen atoms from specific organic substrates in an oxidative reaction that produces hydrogen peroxide.
RH2
+ O2 
R + H2O2
Hydrogen peroxide is very toxic to organic material and is rapidly removed by catalase, which uses it to oxidize other substances including phenols, formic acid, formaldehyde and alcohol.
catalase
H2O2 + R’H2
R’
+ 2H2O
Proliferation of peroxisiomes is an adaptative response to environmental stimuli. It consists of two phases: budding of pre-existing peroxisiomes and growth of the new organelles by importing peroxisomal matrix proteins.
Peroxisomal Proliferation Activation Receptors (PPARs) are transcription factors that ultimately increase synthesis of peroxisomal enzymes. Environmental stimuli increases the synthesis of PPARs in the cytoplasm. A nuclear localization signal (NLS) allows the PPARs to enter the nucleus and activate the genes that encode for oxidative enzymes.
There are two ways for peroxisomal enzymes to enter the peroxisome. In one method, catalase combines with heme iron to form a catalase tetramer. Each of the units has a carboxylic tail with a Ser-Lys-Leu (SKL) signal, which is recognized by a receptor called PTS1R. The receptor binds the SKL signal and brings the tetramer to the membrane of the peroxisome, allowing the complex to interact with the Pex14p receptor. The new complex translocates through the peroxisomal membrane. Once inside, PTS1R lets go of the signal and returns to the cytoplasm.
Another method uses a signal that some enzymes have near the amino terminus. This sequence is bound and carried by PTS2R, which in turn binds the Pex14p receptor and goes thru the peroxisomal membrane. Once in the lumen, PRS2R will cleave the amino terminus signal and leave the oxidative enzyme.
There are at least 8 genes involved in the transport of oxidative enzymes into the peroxisome. When mutation of these genes exists, the person develops Zellweger syndrome. In group 1 of the syndrome, almost all of the peroxisiomes are completely depleted of enzymes. On group 2, some of the transport mechanisms are mutated, so peroxisiomes will have lesser enzymes than normal. In adrenoleukodystrophia (ALD) group 3 disorder, there is only one functional peroxisomal enzyme: fatty acetyl CoA synthetase, but the transport protein is defective so it never gets into the organelle.
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Mitochondria
The mitochondria is a 0.5 to 2.0 mm organelle of variable morphology, sometime branched. It contains two different membranes.
The outer membrane is about 50% protein and 50% lipids, and its proteins mediate the transfer of molecules up to 10 kD. The inner membrane is 80% protein and less permeable. Its surface area is increased by infoldings known as cristae. Its main function is the generation of ATP by oxidative phosphorylation.
Mitochondria are plentiful in cells with high energy demands, and are organized at sites of ATP utilization. In skeletal and cardiac muscle, mitochondria are organized along myofibrils. In spermatozoa, they are organized around the flagella.
The space enclosed by the inner membrane, the matrix, is moderately dense and contains strands of circulars DNA, ribosomes and small granules. Mitochondria are able to code for part of their proteins with these tools.
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