The Golgi Apparatus consists of one or more stacks of disc shaped cisternae, each stack organized as a series of at least 3 functionally distinct compartments, called, cis, medial, and trans cisternae. Proteins and lipids move through the Golgi stack in the cis to trans direction either by vesicular transport or by progressive maturation of the cis cisternae that migrate continuously through the stack. 

Import of Proteins from the ER: A specific GEF embedded in the ER membrane binds to cytosolic Sar 1(which is a coat recruitment monomeric GPAse protein (not a trimeric GTP-binding protein (G protein)) causing Sar1 to release its GDP and bind GTP which exposes a fatty acid tail on Sar1 that inserts into the lipid bilayer of the ER membrane. Membrane bound active SarI-GTP now recruits COPII subunits to the membrane which causes the membrane to form a bud and fuse off. Hydrolysis of bound GTP to GDP then causes Sar1 which is part of the COP11 coated vesicle to once again change its conformation so that its fatty acid tail pops out of the membrane, causing the vesicle’s coat to disassemble, thereby releases the vesicle.

Complementary sets of vesicle SNARES called v-SNAREs and target membrane SNAREs called t-SNAREscontribute to the selectivity of transport vesicle docking and fusion. The vSNARES are packaged with the coat vesicles from the ER donor membrane and bind to complementary tSNAREs in the target membrane of the Golgi. When a v-SNARE interacts with a t-SNARE, the helical domains of one wrap around the domains of the other. These complexes must be disassembled before the SNAREs can mediate new rounds of transport. An ATPase called NSF uses ATP to unravel the complexes. 

Rab proteins are also important for the specificity of vesicular transport. As with the coat recruitment GPAse, Sar1, Rab is also a monomeric GPAse. A GEF in the donor membrane recognizes a specific Rab protein and induces it to exchange GDP for GTP which alters the conformation of the Rab protein, exposing its covalently attached lipid group which helps anchor the protein in the membrane. The Rab-GTP binds to Rab effector proteins which are present on the target membrane thereby helping the vesicle to dock and facilitating the pairing of the appropriate v-SNAREs and t-SNAREs. After vesicle fusing, Rab hydrolyzes its bound GTP releasing Rab-GDP into the cytosol which it can be reused.

After transport vesicles bud from the ER and shed their coat, they begin to fuse with each other to form vesicular tubular clusters. This fusion is called homotypic fusion to distinguish it from heterotypic fusion where a membrane of one comaprtment fuses with the membrane of a different compartment. As with heterotypic fusion, homotypic fusion requires a set of matching SNAREs which are contributed, however, by both membranes this time. As soon as the vesicular clusters form, they begin budding off vesicles of their own which are COPI coated. ARF proteins are responsible for COPI coat assembly (ARF will also be important in clathrin coate assembly) These vesicles carry back to the ER resident proteins that have escaped as well as other proteins that are turned to participate in the ER budding reaction again. The retrieval pathway depends on certain . These retention signals on ER membrane proteins can interact directly with the COPI coat, but retention signals on soluble ER resident proteins (like BiP) must bind to specific receptor proteins like the KDEL receptor which binds to the retention sequence and packages the protein into COPI coated vesicles. This KDEL receptor has a high affinity for escaped ER resident proteins in the vesicular tubular clusters as well as in the Golgi apparatus and low affinity for the sequence in the ER where it unloads its cargo. This affinity is governed by the pH range which are more acidic in the clusters and Golgi as compared to the neutral pH in the ER. 

In vesicular transport, membrane enclosed transport vesicles bud off from one compartment (donor) and fuse with another (target) compartment. In the process, the orientation of both proteins and lipids in the donor compartment membrane are preserved in the target compartment membrane. Thus membrane proteins which face the cytosol will continue to face the cytosol of the cell in the target membrane. Thus if an amino terminus of the protein is in the lumen of the ER, it will face outside the cell after it is transported through the CM.

Protein Modification: A modification that occurs in the Golgi Apparatus is the linkage of an oligosaccaride to the hydroxyl group on the side chain of a serine or threonine in what is called an O-linked oligosaccharide. 

In addition, further modifications occur in the Golgi to the attached to many proteins in the ER. In the processing pathway in the Golgi, the bond between two N-acetylglucosamines becomes resistant to attack by a highly specific endoglycosidase (Endo H). Since all later structures after this point in the pathway are also Endo H-resistant and later pathways after this point occurs in the Golgi, treatment with this enzyme is widely used to distinguish complex oligosaccharides (which have more sugars added in the Golgi). On an agarose gel, you will see a slower running band corresponding to the product made in the Golgi which is endo resistent.

Export: There are 3 main pathways of protein export from the Golgi apparatus. (1) The first pathway is a default and constitutive pathway where proteins are delivered from the trans Golgi network to the plasma membrane unless they are diverted into other pathways or retained in the Golgi. (2) Some specialized cells have a regulated secretory pathway, however, where substances are initially stored in secretory vesicles for release when some signal occurs. Nerve cells, for example, make neurotransmitters and package them into vesicles in the cell body where the ribosomes, ER, and Goli are located. These vesicles travel along the axon to the nerve terminals where they remain until the cell receives a signal such as a hormone to fuse. The resulting activation of receptors by the signals typically increases the concentration of free Ca2+ in the cytosol. (3) As seen, proteins with the mannose M6P marker are diverted to lysosomes via late endosomes in clathrin coated transport vesicles.

The extracellular matrix is composed of a variety of proteins and polysaccharides that are secreted locally and assembled into an organized meshwork in close association with the surface of the cells that produce it. In most connective tissues, the matrix macromolecules are secreted largely by cells called fibroblasts such as the chondroblasts in cartilage and osteoblasts in bone. At the interface between an epithelium and connective tissue, the matrix forms a basal lamina which are flexible, thin mats of specialized EM that underlie all epithelia cell sheets and tubes. Although the precise composition varies from tissue to tissue, most basal laminae contain (1) Type IV collagen, (2) the large heparan sulfate proteoglycan perlecan, (3) the glycoproteins laminin and (4) nidogen. 

The EM was once thought to serve mainly as as just a scaffold but it is now recognized to have an active role in a cell’s development, migration, proliferation, shape and function. Two main classes of extracellular macromolecules that make up the matrix can be classified as either (I) soluble or (II) insoluble:

Souble Extracellular macromolecules of the EM

Glycosaminoglycans (GAGs) hich include soluble polysaccharid chains which are usually found covalently linked to protein in the form of proteoglycans.  GAGs are unbranched chains of repeating disaccharide units. They are called GAGs because one of the 2 sugars in the repeating disaccharide is always an amino sugar which in most cases is sulfunated making GAGs highly negatively charged (anionic). This property means that such GAGs can be easily purified with  and then eluted with a Na+gradient. Except for hyaluronan, all GAGs are found covalently attached to protein in the form of proteoglycans. The GAGs are linked to a tetrasaccharide which is linked to a  side chain on the core protein.  4 main groups of GAGs distinguished according to their sugars are the following: 

Hyaluronans differs from the other GAGs in that it contains no sulfated sugars, chain length is enormous, is generally not linked covalently to any core protein, and is spun out directly from the cell surface by an enzyme complex embedded in the plasma membrane whereas the other GAGs are synthesized inside the cell and released by exocytosis. Hylauronans are important as a space filler during embryonic development where it can be used to force a change in the shape of a structure as it expands with water to occupy a large volume. Hyaluronan has also been shown to play a significant role in the response to non-infectious lung injury. Short-fragment hyaluronan (sHA) is release in the lung after steril injury and can modify the tissue response to injury. Hyaluronan has also been idnetified in airway secretions from asthmatics and high molecule weight hyluronan can attentuate the cronchoconstrictive response in exercise-induced asthma.

Chondrotin sulfate and dermatan sulfate 

Heparan sulfate and 

Keratan sulfate

Proteoglycans are easily distinguished from other glycoproteins by their large weight due to their long, unbranched GAG chains. Some proteoglycans include (1) aggrecan which is huge with over 100 GAG chains (like chondroitin sulfate) is a major component of cartilage. Aggrecan can even assemble with hyaluronan in the extracellular space to form aggregates that are as big as a bacterium. Aggreagate formation is mediated via the proteoglycan tandem repeat loops in the N-terminal G1 domain which bind to decasaccharid units within the polymeric HA. This binding is further stabilized by link protein, which binds to form stable trimeric complexes. Aggrecan is a chondroitin sulfate and keratan sulfate-bearing proteoglycan. A major feature of the pathology of arthritis is the gradual loss of aggrecan from the carilage matrix. 

Decorin which is much smaller with 1 GAG chain (chondroitin sulfate or dermatan sulfate) and can bind to TGFbeta and inhibit its activity. (3) syndecans (GAG chains are chondroitin sulfate & heparan sulfate) which are located on the surface of many types of cells where they serve as receptors for matrix proteins. Syndecans can be found in  where they modulate function.

Insouble Extracellular macromolecules of the EM

Collagen, which are the most abundant proteins in mammals constituting 25% of their total protein mass. In contrast to GAGs which resist compressive forces, collagen fibrils form structures that resist tensile forces. The fibrils are organized in different ways in different tissues. The connective tissue cells themselves determine the arrangement of the collagen fibrils as for example by exerting tension on the matrix. The primary feature of a collagen molecule is its long, stiff triple stranded helical structure in which 3 collagen polypeptide alpha chains are wound around one another. (which because of its ring structure stabilizes the helical conformation in each alpha chain) and (which is regularly spaced at every 3rd residue and due to being the smallest amino acid (having only a H atom as a side chain) can fit into the helix are abundant in collagen. Individual collagen polypeptide chains are synthesized on membrane bound ribosomes and injected into the  as precursors called pro-? chains. In the lumen of the ER, selected and are hydroxylated which are thought to help stabilize the triple stranded helix. Conditions that prevent proline hydroxylation such as a deficiency of ascorbic acid (vitamin C) causes scurvy which loosens the matrix in blood vessels and teeth. After secretion from the cell, the propeptides found at both the collagen N and C terminal ends are removed by specific proteolytic enzymes. The collagen molecules then assemble in the extracellular space to form much larger collagen fibrils. The fibrils are then strengthed by the formation of covalent cross-links between lysine residues. The main types of collagen found in connective tissues are Type I or fibrillar collagens which is the principal collagen of , Type IX or fibril associated collagen which link fibrils with one another to form fiber, and Type IV or network forming collagens that constitutes a major part of the basal lamina.

Elastin, which like collagen is unusually rich in proline and glycine but unlike collagen is not glycosylated and contains some hydroxyproline but no hydroxylysine. Elastin is the dominant matrix protein of arteries comprising 50% of the dry weight of the aorta. Elastin resists not only tensile forces but also has the ability to recoil like a rubber band.

Fibronectin: 

The EXM of some cells is attached to the plasma membrane by the glycoprotein fibronectin. Fibronectin molecuels bind not only to ECM glycoproteins but also to proteins called integrins. Integrins are an integral part of the plasma membrane, extending into the cytoplasma, where they are attached to the microfilaments and intermediate filaments of the cysotskeleton. Linking the ECM and cytoskleton, inegrins allow the ECM to influence cell behavior in important ways. They can alter gene expression and cell migration patterns by a combiantion of mechanical and chemical signaling pathways. 

Fibronectin, a large glycoprotein, is a dimer composed of two very large subunits joined by disulfide bonds near the C-termini. Each chain is folded into domains specialized for binding to a particular molecule (i.e., collagen or heparin) or to a cell. Transcription produces a single large RNA molecule that can be alternatively spliced to produce various isoforms of fibronectin. The main isoform, the type III fibronectin repeat is the most common. A specific RGD (Arg-Gly-Asp) sequence is found in the cell binding domain of one of the type III repeats. Even very short peptides contain this RGD sequence can compete with fibronectin for the binding site on cells, thereby inhibiting the attachment of the cells to a fibronectin matrix. Fibronectin is important not only for cell adhesion to the matrix but also for guiding cell migrations in vertebrate embryos. Large amounts of fibronectin, for example, are found along the pathway followed by migrating prospective mesodermal cells during gastrulation.

laminin is one of the proteins that makes up the basal lamina.

Extracellular proteolytic enzymes (proteases)

The regulated turnover of extracellular matrix macromolecules is important in various processes like when cells migrate through a basal lamina. For example, white blood cells migrate across the BL of a blood vessel into tissues in response to infection. In this process, matrix components such as collagen, laminin and fibronectin are degraded by extracellular proteolytic enzymes (proteases) that are secreted locally by cells. These proteins belong to 1 of two of the following classes:

matrix metalloproteases depend on bound Ca2+ or Zn2+ for activity

serine proteases have a highly reactive serine in their active site. Many proteases are secreted as inactive precursors that can be activated locally when needed. For example, plasminogen is an inactive protease precursor in the blood that is cleaved locally by other proteases called plasminogen activators to yield the active serine protease plasmin which helps break up blood clotes. Tissue type plasminogen activator (tPA) is often given to patients who have had thrombotic stroke to help dissolve the arterial clot.

Calthrin-coated vesicles: The major protein component is clathrin. A second major coat protein of clathrin coated vesicles is adaptin which is required to bind the clathrin coat to the membrane and to trap various transmembrane proteins including transmembrane receptors (called “cargo receptors)” that capture soluble cargo molecules. Clathrin coated vesicles budding from different membranes use different adaptins which are specific for different cargo receptors. 

As a clathrin coated bud grows, soluble cytoplasmic proteins, like dynamin (which is a GTPase), assemble a ring around the neck of each bud. Dynamin recuits other proteins and assist in bending the membrane. 

Once the vesicle is released from the membrane, the clathrin coat is lost. A chaperone protein of the hsp70 family functions as an uncoating ATPase, using the energy of ATP hydrolysis to peel off the coat.

The ER is organized into many branching tubules and flattened sacs extending throughout the cytosol. Its membrane usually constitutes more than 1/2 of the total membrane of an average animal cell. The ER membrane encloses a single internal space called the ER lumen.

In contrast to the import of proteins into mitochondria, nuclei and peroxisomes, import into the ER is a co-translational process in that import begins before the polypeptide chain is completely synthesized. Since one end of the protein is typically translocated into the ER as the rest of the polypeptide chain is being made, the protein is never released into the cytosol. The ribosome that is synthesizing the protein is directly attached to the ER membrane in regions called rough ER. This protection from the cytosol means that proteins bound for the ER do not need chaperone proteins to keep the protein unfolded. Only those mRNA molecules that encode proteins with an ER signal sequence bind to rough ER. Other mRNAs without the sequence remain free in the cytosol. Individual ribosomal subunits are thought to move randomly between these 2 populations of mRNA molecules. 

Rough ER can easily be separated from ER which do not have ribosomes bound (called “smooth ER”) because the rough ER have a higher density and thus float nearer to the bottom of a tube after centrifugation. The main cell type in the liver, the hepatocyte has abundant smooth ER which is the principal site of production of lipoprotein particles which carry lipids via the bloodstream to the body. The enzymes that synthesize these lipoproteins are in the membrane of the smooth ER. In addition, there are enzymes such as the cytochrome P450 family that catalyze a series of reactions which detoxify harmful substances. When the drug phenobarbital is given, the smooth ER doubles in surface area within a few days due to the synthesis of detoxification enzymes.

An ER signal sequence (which varies greatly in amino acid sequence) which emerges from the ribosome is bound by a signal recognition particle (SRP) that has a large hydrophobic pocket that can accommodate many different hydrophobic signal sequences. This binding by the SRP causes a pause in protein synthesis which gives the ribosome time to bind to the ER membrane. The SRP-ribosome complex binds to a SRP receptor which is on the cytosolic surface of the rough ER membrane. The SRP and SRP receptor are then released and the growing polypeptide chain is transferred across the membrane through a protein translocator having a water filled pore called the Sec61 complex. It is thought that a lumenal ER protein serves as a plug when no ribosome is bound to the complex.

Translocation of proteins which are not translated by ribosomes bound to the ER is similar to that described above except that a hsp70 like chaperone protein called BiP (for “binding protein”) on the lumenal side of the ER membrane binds the polypeptide chain as it emerges from the pore into the ER lumen. Unidirectional translocation is driven by cycles of BiP binding and release as described for the hsp70 proteins that use a ratchet like mechanism in import of proteins to mitochondria.

As with mitochondria, the ER signal sequence is cleaved once the protein crosses the membrane by a signal peptidase on the lumenal side. A separate adjacent cleavage site to the N terminal ER signal sequence signals cleavage. 

The translocation process for proteins that are to remain in the ER membrane is similar to what has been said above except for some modifications. For a transmembrane protein which will end up having its C terminus facing the cytosol and its amino terminus facing the lumen, an additional hydrophobic segment called a stop-transfer signal in the polypeptide chain stops the transfer process and anchors the protein in the membrane. In other mechanisms of integration of a single pass membrane what is important is not a stop transfer signal but rather the location of the ER signal sequence. This sequence is not at the N-terminal side of the protein but is rather found at an internal site. Internal start transfer sequences can bind to the translocation apparatus in either of 2 orientations depending on whether more positively charged amino acids proceed or follow the start sequence. In one orientation, the C terminus will end up facing the lumen side and in the other orientation it will end up facing the cytosolic side of the ER membrane. In multipass transmembrane proteins, the polypeptide chain passes back and forth repeatedly across the lipid bilayer. It is thought that an internal signal sequence serves as a start transfer signal in these proteins to initiate translocation, which continues until a stop transfer sequence is reached. In more complex multi pass membranes a second start signal reinitiates translocation until the next sop transfer is found. 

Some proteins such as the BiP protein discussed above contain an ER retention signal (a KDEL sequence in this case for Lys-Asp-Glu-Leu ) on their C terminus that is responsible for retaining the protein. Many other proteins in the lumen of the ER are destined for other organelles. 

Modification of Proteins in the ER: A single species of asparagine N linked oligosaccharid is attached to many proteins in the ER and then trimmed.

N-linked glycosylation of proteins in the lumen is necessary for the proper folding of these proteins. An ER chaperone protein called calnexin binds to incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides which traps the protein in the ER. Removal of the glucose by a glucosidasereleases the protein from calnexin. An enzyme called glucosyl transferase then determines whether the protein is folded properly. If it is still incompletely folded, the enzyme adds a new glucose to the N-linked oligosaccharide which starts the cycle with calnexin again. This occurs until the protein is property folded whereupon it exits from the ER. If a protein simply can not fold properly it is exported back into the cytosol where it will be ubiquitylated. The chaperone protein BiP (discussed above) also may retain misfolded proteins. 

An accumulation of misfolded proteins in the ER triggers an unfolded protein response which includes an increased transcription of genes encoding ER chaperones and enzymes involved in ER protein degradation. This pathway is quite remarkable in that a transmembrane protein kinase in the ER is activated by misfolded proteins which causes autophosphrylation and activation of an endoribonuclease domain that cleaves a cytosolic RNA molecule excising an intron. These exons are joined by an RNA ligase which is then translated on ribosomes to produce a gene regulatory protein that migrates to the nucleus and activates transcription of genes.

The retention of unfolded proteins can be extensive. In the case of newly synthesized subunits of the T cell receptor, for example, more than 90% of the proteins are retained. Sometimes the process goes ary causing disease. For example, cystic fibrosis is due to an active protein that is discarded rather than because a mutation inactivates the protein.

Rough ER:

Proteins synthesized on teh surface of the rough EF (RER) are destinged to be exported form teh cell, sent to lysosomes or vauoles or embedded in the plasma membrane. These proteins enter the cisternal space as a first step in the pathway that will sort proteins to their eventual destinations. 

Smooth ER:

Regions of the ER with relatively few bound ribosomes are refered to as smooth ER (SER). The sturcture of the SER ranges form a network of tubules, to flattened sacs, to higher order tubular arrays. The membranes of the SER contain many embedded enzymes, involved in the synthesis of a vareity of carbohydrates and lipids. Seroid hormones are also synthesized in the SER. The majority of membrane lipids are assembled in the SER and then sent to whatever parts of the cell need membrane components. 

Functions: 

Intracellular Ca2+ storage: An important function of the SER is to store intracellular Ca2+. This keeps the cytoplasmic level low, allowing Ca2+ to be used as a signaling molecule. In muscle cells, for example, Ca2+, is used to trigger muscle contraction. In other cells, Ca2+ release from SER stores is involved in diverse signaling pathway. 

Modify substances to make them less toxic: Another role of the SER is to modify foreign substances to make them less toxic. In the liver, enzymes in the SER carry out this detoxificaiton. This can remove substances that we have taken for a therapetic reason, for enstance, penicillin. Thus, relatively high doses of some drugs are presribed to offset our body’s efforts to remove them. Liver cells have extnsive SER with enzymes that can process a vareity of substances by chemically modifying them. 

Extracellular Vesicles (EVs): See also Drug delivery using exosomes

Mammalian cells normally secrete two types of EVs, smaller vesicles of about 30-200 nm that are commonly referred to as exosomes, and larger vesciels (typically >300 m) that are commonly referred to as microvesciels. (Gould, “Exosome-medaited mRNA delivery in vivo is safe adn can be used to induce SARS-CoV-2 immunity” J. Biiological Chemistry, 2021).

Exosomes: are hihgly enirched in specific substes of proteins, RNAs, and lipids, providing strong evdience that they are generated by an active sorting and vesciel biogenesis pathway. Once released into the extracellular milieu, exosomes can transmit signals and molecules to other cells. Consistent with their natural ability to trasfer RNAs between distinct cells, several groups have demonstrated that these bionormal nanovesicles can be laoded with synthetic small RNAs. In addition, RNA loaded exosomes can be used to deliver anti-cancer RNAs to and into tumors and tumor cells, inhibiting the expression of the target mRNAs, suppressing tumor growth and extending the lifespan of tumor-bearing animals. (Gould, “Exosome-medaited mRNA delivery in vivo is safe adn can be used to induce SARS-CoV-2 immunity” J. Biiological Chemistry, 2021).ureExo exosome isolation kit and ExoCap exosome isolation kit. (Ibrahim, US 2021/0032598). 

Exosomes can be prepared uisng a commercial kit such as ExoSpin Exosome Purificaiton Kit, Invitrogen Total Exosome Purificaiton kit, P

Microvesciels: have a molecule composition that is closer to that of the cell, and there is little if any evidence for a selective biogenesis pthway for this much alrger class of EVs. (Gould, “Exosome-medaited mRNA delivery in vivo is safe adn can be used to induce SARS-CoV-2 immunity” J. Biiological Chemistry, 2021).

See also cell development.

Asexual Reproduction

Organisms can reproduce without sex. For example, there are species of lizards that consist only of females and reproduce without matting. Such asexual reproduction gives rise to offspring that are genetically identical to the parent.

Most yeast reproduce asexually be cell fission or budding, when a smaller cell forms form a larger one. Soemtimes two yeast cells of opposite mating type fuse to form a dikaryon. This cell may tehn function as an ascus, with karyogamy followed by meiosis. The resulting ascospores germinate into new asexually reproducing haploid yeast cells.

Sexual Reproduction:

In contrast, sexual reproduction leads to offspring that differ genetically from both their parents. Most animals reproduce sexually. Animal egss, which are not mobile, are much larger than the small, usually flgellated sperm. In animals, cells formed in meiosis funciton as gametes. These haploid cells do not divide by mitosis first, as they do in plants and fungi, but rather fuse directly with each other to form the zygote. 

In sexual reproduction, genomes mix when two haploid cells (each carrying a single set of chromosomes) fuse to form diploid cells (each carrying a double set of chromosomes). Later, new haploid cells are generated when a descendant of this diploid cell divides by the process of . During meoisis, the chromosomes of the double chromosome set exchange DNA by genetic recombination before being sorted out in new combinations into single chromosome sets. 

Haploid cells that are specialized for sexual fusion are called gametes. (Gametes are produced by meiosis.) Typically two types of gametes are formed: one is large and nonmotile and is called the egg (or ovum) and the other is small and motile and is called the sperm (or spermatozoon). During the diploid phase that follows the fusion of gametes, the cells proliferate and diversify to form an embryo. 

In all vertebrate embryos, certain cells are singled out early in development as progenitors of the gametes. These primordia germ cells migrate to the developing gonads, which will form the ovaries in females and the testes in males. The determination as to whether these primordial germ cells will develop into eggs or sperm depends not on their own makeup but rather on whether the organ to which they have migrated develops into an ovary or a testes. This in turn depends on the sperm that fertilized the egg. Eggs have a single X chromosome, whereas the sperm can have either an X or a Y. A sperm that carries a Y chromosome instead of an X chromosome will induce somatic cells of the developing gonads into a testes.

Developmental Pathway to Testes

The critical gene on the Y chromosome that has a testes determining function is called Sry (“sex-determining region of Y). The  Sry gene on the Y chromosome contains a TDF region which results in differentiation of the testes. Without this region, the female gonad will form instead.

Sry is expressed only in a subset of the somatic cells of the developing gonad, and it causes these cells to differentiate into sertoli cells which are the main type of supporting cells found in the testes. Sertolli cells have a number of functions such as the following:

• support developing germ cells as they differentiate from spermatogonia to sperm such as by producing lactate. This is important since the blood testis barrier prevents nutritional components from reaching the later stages of the germ cells. This need is provided by sertoli cells;
• divides the seminiferous tubules into basal and adluminal compartments by sertoli-sertoli cell tight junctions. 
• secrete anti-Mullerian hormone which suppresses the development of the female reproductive tract by causing the mullerian duct to regress;
• induce other somatic cells in the developing gonad to become leydig cells. Leydig cells secrete the male sex hormone testosterone. Testosterone, as other steroids, is derived from cholesterol and is stimulated by the action of luteinizing hormone (LH) (there is a LH receptor on the leydig cell) from the anterior pituitary. Testosterone is responsible for inducing the development of many ducts and accessory glands.

Spermatogenesis in the Male

Spermatogenesis is the process of differentiation from a diploid spermatogonia to a haploid spermatozoa through the process of meiosis and differentiation.

In spermatogenesis, immature germ cells called spermatogonia (singular, spermatogonium) proliferate continuously by mitosis around the outer edge of the seminiferous tubules next to the basal lamina which surrounds the seminiferous tubule. Some of the daughter cells stop proliferating and differentiate into primary spermatocytes. The process of developing primary spermatocytes from spermatogonia is referred to as spermatocytogenesis. 

Primary spermatocytes then proceed with  to produce two secondary spermatocyteseach containing 22 duplicated autosomal chromosomes and either a duplicated X or a duplicated Y chromosome. These two secondary spermatocytes then proceed through to produce four spermatids each with a haploid number of single chromosomes. These haploid spermatides then undergo morphological differentiation into spermatozoa which escape into the lumen of the seminiferous tubule. The process of development of spermatozoa from spermatids is referred to as spermiation.

Developmental Pathway to Ovary

In the absence of Sry, the genital ridge develops into an ovary. The supporting cells become follicle cellsinstead of Sertoli cells. Follicle cells are arranged as an epithelial layer around the oocyte to which they are connected by gap junctions which permit the exchange of small molecules. 

Other somatic cells become theca cells instead of Leydig cells and secrete at the beginning of puberty the female sex hormone estrogen instead of testosterone. The primordial germ cells develop into eggs instead of sperm. 

Oogenesis

Oogenesis is the development of an oocyte (egg). In this process, primordial germ cells migrate to the forming gonad which then proliferates by mitosis before differentiating into primary oocytes where the first  begins (usually before birth in mammals). In mammals, primary oocytes remain arrested in prophase of meiotic division I until the female becomes sexually mature where under the influence of hormones a small number of primary oocytes periodically mature to become secondary oocytes, completing division I. At ovulation, the arrested secondary oocyte is released from the ovary and undergoes a rapid maturation step that transforms it into an egg that is prepared for fertilization. If fertilization occurs, the egg is stimulated to complete meiosis. 

Prior to ovulation, follicular cells proliferate and secrete estrogen under FSH stimulation. There is also a surge in LH which induces ovulation. After ovulation, there is synthesis of progesterone as well as estrogen which inhibit LH and FSH secretion. 

Fertilization

Once released, egg and sperm will die within minutes/hours unless they find each other and fuse in the process of fertilization. To become competent to migrate through the layer of follicle cells and then bind to and cross the zona pellucida (egg coat) the sperm must become competent by conditions in the female reproductive track in a process called capacitation. This requires 5-6 hours in humans and is triggered by bicarbonate ions in the vagina which enter the sperm and activate a soluble  enzyme in the cytosol. Capacitation involves alterations in membrane characteristics and increased

On binding to the zona, the sperm is induced to undergo an acrosome reaction in which the contents (proteolytic enzymes) of the acrosomal vesicle at the head of the sperm are released which allow the sperm to penetrate the zona. This release is triggered by a glycoprotein on the oocyte called ZP3. 

When the sperm fuses with the ova, it causes an increase in calcium ions which leads to the initiation of something called the cortical reaction or the release of cortical granules from the egg by exocytosis. The contents of the cortical granules include various enzymes that change the structure of the zona pellucida which hardens it so that sperm no longer binds to it. It thus provides a block to fertilization of more than one sperm. 

Zygote: (fertilized egg): has the capability of giving rise to all the kida of cells in an animal’s body. It is accordingly totipotent. Fertilization is completed when the two haploid nuclei come together and combine their chromosomes into a single diploid nucleus. In humans the sperm contributes a centriole to the fertilized egg which the egg lacks. The centriole replicates and helps organize the assembly of the first mitotic spindle in the fertilized egg (zygote). Fertilization is the start of  in which the zygote develops into a new individual.

Fertilization in Particular types of Organisms:

Amphibians:

Amphibians’ eggs must be laid in water or a moist setting to avoid drying out.

–Frogs and toads: return to water to reproduce, laying their eggs directly in water. Their eggs lack watergith external membrane and would dry out quickly on land. Eggs are fetrilized externally and hatch into swimming larval forms called tadpoles. Tadpoles live in the water, where they geenrally feed on algae. After considerable growth, the body of the tadpole gradually undergoes metamorphosis into that of an adult frog.

Reptiles:

Reptiles are a higly successful group. There are mroe living species of snakes and lizard than there are of mammals. Reptiles occur world wide except in the coldest regions, wehre it is impossible for ectotherms to survive.

Reptiles do not practice external fertilization as most amphibians do. Sperm would be unable to penetrate the membrane barreris protecting the egg. Instead, the male places sperm inside the female, where sperm fetilizes the egg before the protective membranes are formed. This is called internal fertilization.

Although marine turtles spend their lives at sea, they must return to land to lay their eggs. Many species migrate long distances to do this. Atlantic green turtiles migrate from their feeding grounds off the coast of Brazil to the middle of the South Atlantic, a distinace of more than 2000 im, to lay their eggs on the same beaches wehre they were hatched themselves.

One of the major features of vertebrates which do not have any counterpart in say Drosophila is the formation of a notochord. A picture of this formation is illustrated with the frog, zenopus laevis. Here, the cells that were near the  involute first, turning inward and then moving up toward the animal pole to form the most anterior part of the gut. As they near the  , these leading  cells will signal to the overlying   to define the anterior extremity of the head. A slender rod of cells with endoderm below it, ectoderm above it and mesoderm on either side is the notochord. 

In a sheet of ectoderm overlying the notochord other cell movements occur in a process known as neurulation. First, a broad central region of ectoderm, called the neural plate, thickens, rolls up into a tube, and pinches off from the rest of the cell sheet. The tube created is called the neural tube and it will give rise to the central nervous system (brain and spinal cord). Meanwhile a sheet on the dorsal side of the neural tube, called the neural crest migrates away from the surface of the neural tube and gives rise to a diverse set of cell types of the peripheral nervous system.

The neural tube consists initially of a single layered epithelium. The epithelia cells are progenitors of and cells. The locations where neural cells are born are called germinal zones. Prenatally, this zone is the ventricular zone/neural tube. Postnatally, the zones are the subventricular zone/Olfactory bulb (OB) and hippocampus. 

Newborn neurons go through their final division close to the inner surface of the neural tube and then often migrate outward by crawling along radial  before sending out axons and dendrites. The first  born neurons settle closest to their birthplace, while neurons born later crawl past them to settle farther out. Thus successive generations of neurons occupy different layers in the cortex.

The long held belief that neuron generation is restricted to the embryonic period has been altered with evidence that new neurons continue to be added to certain regions like the hippocampus and olfactory bulb of the adult vertebrate brain. This generation of new neurons or neurogenesis has been shown in human postmortem brain tissue treated with the thymidine analog, bromodeoxyuridine (BrdU) that labels DNA during S phase. Using immunofluorescent labeling for BrdU and neuronal markers, new neurons as defined by these markers are generated from dividing progenitor cells in the adult human hippocampus. Enhancers of neurogenesis include physical activity and antidepressants. Inhibitors of such neurogenesis include aging and stress.

Principles of Development as Learned through the Fly

Much of the basic principles of has been learned through the fruit fly, Drosophila melanogaster. Some of these basic principles are illustrated here:

• Importance of   and signaling molecules in development: 

One example of the importance of transcription factors are the proteins bicoid and hunchback which are important in determining the anterior part of the body (head and thorax) from the posterior part (abdomen) of the fruit fly. Bicoid mRNA is expressed as a bicoid protein resulting in a protein gradient with the highest concentration of protein on the left side of the embryo. Hunchback DNA is only activated once the amount of bicoid protein passes a certain threshold. This results in a sharp borderline which in the developing embryo from the part where hunchback is not expressed to the part where hunchback is expressed. 

The Hox genes play a very important role in early embryological development. There are 8 Hox genes in the fly and some 39 Hox genes in humans. These genes lie in clusters. In the fly, for example, the genes lie in two clusters, one cluster which controls the differences among thoracic and head segments and the other which controls differences among the abdominal and thoracic segments of the body. All contain a highly conserved homeobox domain. This homeobox domain encodes a small protein that is a transcription factor (binds DNA & regulates gene activity). 

The coding sequences of the 8 HOX genes are interspersed amid a larger quantity of regulatory DNA. This regulatory DNA along with HOX interpretes the multiple items of positional information 

One important signalling pathway in animal development is the pathway activated by Hedgehog proteins. Two transmembrane proteins, patched and Smoothened mediate responses to all Hedgehog proteins. Hedgehog is an extra cellular signaling molecule that binds to patched freeing Smoothened from patched. The way that this works is that in the absence of Hedgehog, a  called Cubitus Interruptus (Ci) is cleaved into a smaller protein that accumulates in the nucleus where it acts as a transcriptional repressor, helping to keep some Hedgehog responsive genes silent. When Hedgehog binds to Patched, this processing is stopped and the uncleaved Ci protein is released from its complex where it enters the nucleus and activates the transcription of Hedgehog target genes. Sonic Hedgehog also can play a role in cancer (it facilitates proliferation by stimulating Ci-coativator complex formation which in turn facilitates activation of cyclins D and E promoters. Inactivating mutations in Patched also allow Ci-coactivator formation as with Hedgehog binding to patched)

importance of   of gene regulatory proteins and importance of noncoding DNA sequences: One example is the Drosophila even-skipped (eve ) gene. At the earliest stage of development where eve is expressed, the embryo is a single giant cell containing multiple nuclei in a common cytoplasm. But this cytoplasm is not uniform. It contains a mixture of gene regulatory proteins that are distributed unevenly along the length of the embryo, thus providing positional information that distinguishes one part from another. The eve gene contains regulatory noncoding DNA sequences that somehow read the concentrations of gene regulatory proteins at each position along the length of the embryo such that eve is expressed in seven stripes positioned precisely along the anterior-posterior axis of the embryo. When a particular regulatory module for say stripe 2 is removed from its normal position upstream of the eve gene, placed in front of a reporter gene (promoter + B-galactosidase gene) and reintroduced into the fly genome, the reporter gene is found to be expressed in precisely the position of stripe 2. How can this be? The regulatory module contains recognition sequences for 2 gene regulatory proteins (Bicoid and Hunchback) that activate eve transcription and 2 (Kruppel and Giant) that repress it. The relative concentrations of these 4 proteins determine whether protein complexes forming at the stripe 2 module turn on transcription of the eve gene. The regulatory unit combines with these 4 proteins to turn on transcription of eve only in those nuclei that are located where the levels of both Bicoid and Hunchback are high and both Kruppel and Giant are absent. This combination of activators and repressors occurs only in one region of the early embryo whereas everywhere else the stripe 2 module is silent.

Embryogenesis starts out with the fertilized egg which divides or cleaves to form many smaller cells (blastomeres) without any change in total mass. The determinants distributed asymmetrically in the egg become partitioned into separate cells. During this period of cleavage, the embryo transforms from a solid sphere of cells into more of a hollow ball, with an internal fluid filled cavity surrounded by cells that cohere to form an epithelial sheet now called a blastula. But then gastrulation occurs which is the transformation of this ball into a structure with a gut. A part of the ectoderm which is the precursor to the epidermis and nervous system becomes tucked into the interior to form endoderm which is the precursor of the gut, lung and liver. Another group of cells move into the space between ectoderm and endoderm to from the mesoderm which is the precursor of muscles and connective tissues.

Gastrulation is just the first of a variety of cell movements that shape the parts of the body. Another extremely important movement is the process of where a broad central region of the ectoderm rolls up into a tube to create the which will form the brain and spinal cord.

Many proteins regulating development, especially pattern formation, are either  or signaling molecules. Cells do not differ because they contain different genes (in fact their genes are usually identical) but rather because cells express different genes. To a large extent, the instructions needed to produce a multicellular animal are contained in the non-coding regulatory DNA that is associated with each gene. The DNA may contain dozens of separate  or  that serve as binding sites for specific complexes of . This selective expression control 4 essential processes by which an embryo develops:

• proliferation: 1 fertilized cell is able to produce millions of other cells;

• differentiation or specialization:groups of cells do different things from other groups of cells. How can such differentiation come about? One way that this might occur, particularly early in development, is by asymmetric cell division in which a set of molecules is divided unequally between 2 daughter cells at the time of cell division. A good example of this is the vertebrate frog, zenopus laevis. The zenopusegg has a lower egg called the vegetal pole and an upper end called the animal pole. The animal and vegetal poles contain different mRNA molecules and other cell components which become allocated to separate cells as the egg cell divides. In the vegetal pole, for example, there is an accumulation of mRNAs coding for the gene regulatory protein VegT as well as some protein components of the Wnt signaling pathway. As a result, the cell that inherits vegetal cytoplasm will produce signals to organize the behavior of adjacent cells and form the gut. Fertilization by the sperm triggers an intracellular movement that gives the egg an additional asymmetry defining a dorsorventral (back to belly) difference. This event leads to a  based transport of the protein Dsh (dishevelled) which is a downstream component of the Wnt signaling pathway toward the future dorsal side. The subcellular region in which Dsh becomes concentrated gives rise to cells that express a dorsal specific set of genes which will generate further signals to organize the dorsoventral axis of the body. 

A more common way where cells differentiate is by exposing cells to signals from neighboring cells. For example, in inductive interaction a group of cells starts out having the same developmental potential but a signal from cells outside the group then drives one or more of the members of the group into a different developmental pathway. Usually the signal is limited in time and space so that only a subset of the cells closest to the source of the signal take on the induced character. The induction can get more complex however. In sequential induction, one group of cells might induce a neighboring group to specialize in a certain way with a subset of its cells closest to the inducer. This subset might then in turn induce its neighbors to specialize.  Induction does not even need to be a positive effect; it can be inhibitory. In lateral induction one or a group of cells specializes in a particular way by delivering a signal to neighboring cells that inhibits them from doing likewise.

A large number of developmental decisions are actually regulated by inhibitors rather than by the primary signal molecule. One such signalling molecule which uses the TGF? signalling pathway is the ligand BMPwith its associated extracellular inhibitors/modulators chordin and noggin. Chordin does not have its own receptor. Instead it is an inhibitor of BMP which is an inducer of the ventral part of the body. Inhibition of BMP by Chordin specifies the dorsal part of the body in the vertebrate frog, zenopus laevis. 

• interactions: the behavior of cells are coordinated with that of their neighbors

• movement: cells are moved and rearranged to form tissues and organs.

Much of what has been said above has come through the study of the .

The cytoplasm of all eukaryotic cells is crisscrossed by a network of protein fibers that supports the shape of the cell and anchors organelles to fixed locations. This network, called the cytoskeleton, is a dynamic system, constantly assembling and disassembling. The cytoskeleton is made up of 3 major elements; microfilaments (“actin”) filaments, intermediate filaments, and microtubules.

Actin filaments (Microfilaments): 

Structure:

Actin filaments are the smallest component of the cytoskeleton ranging in size of about 7 nm in diameter. They are made up of actin and have a plus/barbed fast growing end, and a minus/pointed slow growing end. The addition of actin monomers occurs at the + end and is an ATP dependent process. Polymerization into filamentous f-action occurs when the concentration of globular g-actin is above a critical concentration.

The actin gene originated in the common ancestor of all life on Earth, as evidenced by the fact that bacteria, archaea, and eukaryotes all have actin molecules related structurally and functionally to each other. All eukaryotes have one or more genes for actin, and sequence comparisons have established that they are one of the most conserved gene families, varying by only a few amino acids between algae, amoeba, fungi, and animals. This conservation is attributed to constraints imposed by the interactions of actin with itself to polymerize, with motors and with a large number of regulatory proteins.  Humans have three genes for α-actin (muscles), one gene for β-actin (nonmuscle cells), and two genes for γ-actin (one in some smooth muscles and one in nonmuscle cells). Plants have 10 or more actin genes; some are specialized for reproductive tissues and others for vegetative tissues.(Pollard, “Actin and Actin-binding Proteins” Cold Spring Harbor Perspect Biol, 8/8, 2016). 

Actin is one of the most abundant proteins on Earth and the most abundant protein in many cells, from amoebas to human, often accounting for 10% or more of total protein. Its abundance is topped only by tubulin in brain and keratins in skin. Actin molecules in cells turn over very slowly, on the order of weeks in muscle cells. Pollard, “Actin and Actin-binding Proteins” Cold Spring Harbor Perspect Biol, 8/8, 2016).

Actin is described as having four subdomains. The polypeptide winds from the amino terminus in subdomain 1 to subdomains 2, 3, and 4 and back to subdomain 1 at the carboxyl terminus. ATP binds in a deep cleft, interacting more strongly with subdomains 3 and 4, but also with residues in subdomains 1 and 2. Several proteins bind in a prominent groove between subdomains 1 and 3—and, hence, some call it the “target-binding groove” Actin monomers bind ATP or adenosine diphosphate (ADP) tightly, provided that either Ca2+ or Mg2+ is present in the buffer. One of these divalent cations associates with the β- and γ-phosphates of ATP, stabilizing its interaction with the protein (Pollard, “Actin and Actin-binding Proteins” Cold Spring Harbor Perspect Biol, 8/8, 2016).

Actin-myosin interactions play crucial roles in the generation of cellular force and movement. The molecular mechanism involves structural transitions at the interface between actin and myosin’s catalytic domain, and within myosin’s light chain domain, which contains binding sites for essential (ELC) and regulatory light chains (RLC). (Thomas, ” Actin-Myosin Interaction: Structure, Function and Drug Discovery” Int. J. Mol. Sci. 2018, 19(9))

Functions/mechanisms of action:

Actin filaments are responsible for cellular movements such as contraction, crawling, “pinching” during division, and formation of cellular extensions. Essentially all cell motion is tied to the movement of actin filaments, microtubules, or both.  At the leading edge of a crawling cell, actin filaments rapidly polymerize and their extension forces the edge of the cell forward. Overall forward movement of the cell is acheived through the actin of the protein myosin, which is best known for its role in muscle contraction. Myosin motors along the actin filaments contract, pulling the contents of the cell toward the newly extended front edge. Cells crawl when these steps occur continucrously, with a leading edge extending and stabilizing, and then motors contracting to pull the remaining cell contents along. Receptors on the cell surface can detect molecules otuside the cell and stimulate extension in specific directions, allowing cells to move toward particular targets. 

Actin associated proteins perform many functions in arranging and stabilizing microfilaments including crosslinking (by filamen), bundling (the crosslinking of actin filaments into a parallel array as by ? actininand fimbrin), capping, severing and movement of structures along the fiber.

Contraction depends on the ATP driven sliding of highly organized arrays of actin thin filaments against arrays of myosin thick filaments. 

The zonula adherens/belt desmosome is an achorage junction that has a beltlike distribution and is associated with actin filaments.

Actin monomers polymerize spontaneously under physiological salt conditions with either or both monovalent and divalent cations in the buffer. Cations bind specific sites that promote interactions between subunits in the filament.  Pollard, “Actin and Actin-binding Proteins” Cold Spring Harbor Perspect Biol, 8/8, 2016).

Inhibitors: 

The drug, phalloidin, from the fungus Amanita phaloides (death cap), prevents the depolymerization of actin filaments. cytochalessins bind to the fast growing end preventing further addition of G-actin

Intermediate Filaments: 

Intermediate filamentsare intermediate in size compared to microfilaments and microtubules ranging with a diameter of about 10 nm. There are different types of intermediate filaments depending on the tissue where they are found.

The macula adherens/spot desmosome is an achorage junction which has a spot like distribution and is associated with intermediate filaments.

cytokeratins: are found in epithelia cells.

–Keratin is a filament protein which belongs to the intermediate filament protein family. Keratin fliaments are a major component of the cellular cytoskeleton. 

Keratin proteins are the predominant subtype of IFs in all epithelia and, given a total of 54 conserved functional genes and prtoeins, represents almost three-quarters of the entire IF superfamily in mammals. Kertin includes two types of IF sequences or families: type I, which number 28, and type II, which number 26. 

Keratins are a group of insoluble and filament-forming proteins. They mainly exist in certain epithelial cells of vertebrates. Keratinous materials are made up of cells filled with keratins, while they are the toughest biological materials such as the human hair, wool and horns of mammals and feathers, claws, and beaks of birds and reptiles which usually used for protection, defense, hunting and as armor. Keratins generally exhibit a sophisticated hierarchical structure ranging from nanoscale to centimeter-scale: polypeptide chain structures, intermediated filaments/matrix structures, and lamellar structures. (Fan, “Structure of Keratin” Methods Mel. Biology, 2021). 

vementin in connective tissue.

desmin in  

neurofilaments in axons and dendrites of nerves

nucleur lamins in nuclear lamina

Microtubules: 

Microtubules are formed from protein subunits of tubulin. The tubulin subunit is itself a heterodimer formed from two closely related proteins called alpha-tubulin (exposed at the + end) and beta-tubulin (exposed at the -end) tightly bound by noncovalent bonds. A microtubule is a stiff, hollow cylindrical structure built from 13 parallel protofilaments (long linear strings of subunits joined end to end that associate with one another laterally) They are larger than either microfilaments and intermediate filaments with a diameter of 25 nm. Addition of tubulin is a GTP dependent process unlike with the ATP dependent process of actin. 

Microtubules have one end attached to a single microtubule organizing center (MTOC) called a centrosome which is made up of 2 centrioles near the nucleus. 

Centrioles: are barrel shaped organelles found in the cells of animals and most protists. They occur in pairs, usually located at right angles to each other near the nuclear membranes. The region surrounding the pair is referred to a centrosome. The motor proteins kinesin and dynein move on microtubules and are involved with organelle transport. kinesins typically move from the cell body or centrosome toward the periphery of the cell towards the plus end of the tubule (anterograde) whereas dyneins typically move from the periphery toward the centrosome or minus end of the tubule (retrograde).

All eukaryotic cells must move materials from one place to another in the cytoplasm. One way they do this is using the channels of the ER, but material can also be moved using vesicles loaded with cargo that can move along the cytoskelton. For example, in a nerve cell with an axion that may extend far form the cell body, vesicles can be moved along tracks of microtubules from the cell body to the end of the axon. For components are reqired to do this: (1) a vesicle or organelle that is to be transported, (2) a motor protein that provides the eernergy driven motion, (3) a connector molecule that connects the vesicle to the motor moleule and microtubules on which the viscle will ride. The direction a vescile is moved depends on the type of motor protein involved and the fact that microtubles are organized with their plus ends toward the periphery of the cell. In one case, a protein called kinectin binds vesciles to the motor protein kinesin. Kinsin uses ATP to power its movement toward the cell periphery, dragging the vescile with it.Another set of vesicle proteins, called the dynactin complex, binds veiscles to the motor protein dyein which directs movement in the opposite direction toward the minus end inward toward the cell’s center. Dynein is also involved in the movement of eukaryotic flagella. 

Microtubules and the protein dynein also make up the motility structures, cilia and flagella. flagella are found on sperm and enable these cells to move. Cilia tend to be shorter than flagella. Both flagella and cilia have a distinctive 9 (doublet) + 2 arrangement of microtubules. Microtubules are also necessary in mitosis. 

A number of chemical agents also inhibits microtubule dynamics such as colchicine which binds to tubulin dimers preventing their assembly into microtubules.