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UNIT -5 DEVELOPMENTAL BIOLOGY (GURKEN PROTEIN )VERY VERY IMPORTANT TOPIC

The gurken gene of Drosophila melanogaster encodes a protein with homology to the transforming growth factor alpha (TGF-a) class of signaling molecules.

During Drosophila oogenesis, the gurken gene is transcribed in the cells of the germline. The gurken messenger RNA accumulates in the oocyte and is translated throughout oogenesis. The Gurken protein is transported to the oocyte membrane, and activates the Drosophila epidermal growth factor receptor (Egfr, tyrosine kinase receptor which is expressed on the adjacent follicle cells that surround the oocyte. The activation of this major transmembrane receptor regulates the expression of a number of genes in the follicle cells, and thus initiates a series of events that lead to the correct patterning of both the anteroposterior and the dorsoventral axis of the egg and embryo.

(1). Gurken acts as a ligand that is specific to oogenesis. The Gurken protein consists of an extracellular domain, which harbors a single epidermal growth factor (EGF)-like domain, a transmembrane domain, and a short cytoplasmic domain

 (2). It is homologous to the gene spitz of Drosophila, which acts as ligand of Egfr in the embryo and in imaginal discs.

Early in oogenesis, gurken mRNA is found in the developing oocyte, and the Gurken protein accumulates in the oocyte membrane. At this stage, the oocyte occupies only a small part of the volume of the egg chamber, and therefore only a limited number of follicle cells at the posterior of the egg chamber are in contact with the oocyte membrane. In this group of follicle cells, the Egfr is activated by Gurken protein, which results in the specification of these follicle cells as posterior cells. 

If  Gurken protein is absent, and the follicle cells at the posterior end of the egg chamber are not induced to assume a posterior cell fate. They consequently develop as anterior follicle cells, which results in the production of an egg with two anterior ends. The posterior follicle cells normally send a signal back to the oocyte, which organizes the cytoskeleton of the oocyte and specifies the posterior end of the oocyte. In the mutant egg chambers, no such signal is sent from the follicle cells, and the cytoskeleton of the oocyte is misorganized. RNAs such as bicoid or oskar, which should normally be localized to one end of the egg RNA localization are mislocalized in these mutant oocytes, and the embryos that develop inside such eggs have an abnormal antero-posterior pattern .

In midoogenesis -The oocyte has grown .The gurken RNA accumulates in the region around the oocyte nucleus, and the Gurken protein is now found in a very restricted part of the oocyte membrane, directly overlying the oocyte nucleus. At this stage, Gurken activates the Egfr in the lateral follicle cells that contact the oocyte on the side where the nucleus is situated. Activation of the Egfr in these lateral follicle cells induces them to become dorsal follicle cells. In the absence of gurken signaling, the lateral follicle cells develop into ventral follicle cells . The ventral follicle cells normally regulate the production of a ventral signal that activates the Toll receptor protein on the ventral side of the egg and is responsible for inducing ventral cell fates in the developing embryo. In the strong gurken mutants, therefore, the ventralization of the follicle cell epithelium leads to an overproduction of the ventral signal, and consequently to a ventralized embryo.

Gurken signaling presumably leads to the formation of a broad field of dorsal cell fates, but secondary patterning mechanisms appear to operate to lead to the final, complex pattern of cell fates of the mature egg. In addition, gurken signaling also interacts with signaling through decapentaplegic (dpp), a molecule with homology to TGF-b. In follicle cells that receive both the gurken signal and the dpp signal, formation of dorsal appendages is repressed, and formation of operculum cell fate is induced . The regulation of the embryonic ventral signal by activation of Egfr seems, however, to be independent of the production of dorsal anterior follicle cell fates. In situations where ectopic activation of Egfr in follicle cells is induced in parts of the follicle cell epithelium, embryos result that show only regional dorsalization, corresponding to the region of the follicle cell epithelium where Egfr was ectopically activated .

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IMPORTANT COMPONENTS OF ECM (EXTRACELLULAR MATRIX)

Elastin -This insoluble fibrous macromolecule provides the resilience (stretch) of the skin, vessels, and ligaments by associating with collagen fibers to limit stretching and preventing tearing. It is highly hydrophobic (proline and glycine), is not glycosylated or "hydroxylized". It has many crosslinks at the lysine residues to stablize the fibers. 

Fibrilin -Is bound in fibers of microfibril sheaths. The sheaths are composed primarily of this protein.
The elastic fiber is made of two alternating segments of hydrophobicity and alpha-helical segments (alanine/ lysine --> crosslinks)

Integrins-This glycoprotein is a cell receptor that interacts with the surrounding ECM, providing anchor for the cell to the matrix of the collagen + links to peptidoglycans.This transmembrane cell receptor is made of alpha and beta subunits that function in cell signaling (depend on extracell divalent cations) and the regulation of cell cycle, shape, and motility through LINKING the ECM with the actin cytoskeleton.  Integrins can mediate indirect interactions with the cytoplasmic cytoskeleton through intermediary proteins including talin and vinculin.


Selectins-Carbohydrate binding proteins that bind to glycoproteins from other cells, important in WBC extravasation, the movement of them from inside the capillaries into the tissues

Multiadhesive proteins -

Dthrombospondin,tenascin,vitronectn,nidogen/enactin,van willebrand factor ,laminin and fobronectin.

A. Laminin- This is a large adhesive glycoprotein that is a major component of BASAL LAMINA. It has binding domains specific for other actors in the BL, including type IV collagen, heparin sulfate (perlecan), enactin (nidogen), and integrins. It consists of 3 poly peptide chains: alpha, beta, gamma

B.Fibronectin -
This example of an adhesive glycoprotein is found in most ECM and plasma. It binds to collagen and proteoglycans. Type III module contains RGD sequence for binding receptors. Importantly, it exists as a protein dimer, consisting of two nearly identical monomers linked by a pair of disulfide bonds.
Although Fibronectin is produced from one gene, it has highly diverse isoforms due to this mechanism in pre-mRNA.

Examples:
- Soluble plasma fibronectin - component of blood plasma made by liver
- Insoluble cellular fibronectin - component of ECM.

Proteoglycans -

A.Aggrecan -This is a major macromolecule of cartilage (made of several GAGs). Due to extensive hydration, it creates a gel-like matrix. These proteoglycans require a core of Hyaluronan and it binds to TGF-beta to inhibit ECM synthesis.

B.Decorin-Is a ubiquitous proteoglycan wide spread in ECM binds to type 1 collagen fibrils and can limit their size and binds TGF-beta and sequesters it interaction with cells.

C.Perlecan -Type of proteoglycan, found in basal lamina, structural and filtering function in basal lamina, the glycosaminoglycan chains attached to perlecan are responsible for preventing proteins escaping from the serum to the urine during glomerular filtration, is one of the proteins that can be defective in specific form of muscular dystrophy.

Notes on collagen -

http://dnaofbioscience.blogspot.com/2016/10/collagen.html

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DELTA NOTCH SIGNALLING PATHWAY

The Delta-Notch signalling pathway is a highly conserved cellular signaling 

mechanism that is essential for embryonic development .

Notch genes encode for transmembrane protiens, Delta is an example
 of one of the ligands that bind to the extracellular domain of the
 Notch receptor in Drosophila. Delta is a single-pass transmembrane
 protein ligand that is membrane bound. 

Notch signalling determines cell fate of surounding cells by the process of

 lateral inhibition in both vertbrates and invertebrates. 

Different Notch activation results in different biological alterations in

 gene expression. For example in human adult tissue, activation of Notch1 

corresponds to differentiation of T-cells and self-renewal, which path is 

undertaken is dependent on other environmental factors too.

• It involves proteolytic cleavage to release an intracellular fragment (Nicd) that functions to regulate transcription.
• In the nucleus, Nicd displaces a repression complex and, with the DNA-binding CSL (CBF1, Su(H) and LAG-1) protein, recruits . Additional epigenetic cofactors are implicated and recruitment of kinases and ubiquitin ligases probably contribute to rapid turnover of the activator complex.
• Activity of the receptor is also regulated post-translationally. A number of different auxiliary components are implicated, including several ubiquitin ligases and proteins, such as Numb, that have direct links to the endocytic machinery.
• 

  Notch ligands are transmembrane proteins and they require E3 ubiquitin ligases for their activity. The mechanisms whereby the ligands become competent to signal are not yet known, but probably entail endocytosis. Localization and cleavage of ligands might also contribute to their regulation.

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G Proteins Signal Via Phospholipids

Many GPCRs exert their effects through G proteins that activate the plasma-membrane-bound enzyme phospholipase C-β (PLCβ). The activated GPCR stimulates the plasma-membrane-bound phospholipase C-β (PLCβ) via a G protein called Gq. The α subunit and βγ complex of Gq are both involved in this activation.

 Two second messengers are produced when PI(4,5)P2 is hydrolyzed by activated PLCβ -

1.Inositol 1,4,5-trisphosphate (IP3) IP3 is a water-soluble diffuses through the cytosol and releases Ca2+ from the ER by binding to and opening IP3-gated Ca2+-release channels (IP3 receptors) in the ER membrane. The large electrochemical gradient for Ca2+ across this membrane causes Ca2+ to escape into the cytosol when the release channels are opened.  

2.Diacylglycerol remains embedded in the plasma membrane and, together with phosphatidylserine  and Ca2+, helps to activate protein kinase C (PKC), which is recruited from the cytosol to the cytosolic face of the plasma membrane. 

 The initial rise in cytosolic Ca2+ induced by IP3 alters the PKC so that it translocates from the cytosol to the cytoplasmic face of the plasma membrane. There it is activated by the combination of Ca2+, diacylglycerol, and the negatively charged membrane phospholipid phosphatidylserine). Once activated, PKC phosphorylates target proteins that vary depending on the cell type. 

Diacylglycerol can be further cleaved to release arachidonic acid, which is used in the synthesis of other small lipid signal molecules called eicosanoids. Most vertebrate cell types make eicosanoids, including prostaglandins, which have many biological activities. They participate in pain and inflammatory responses, for example, and many anti-inflammatory drugs (such as aspirin, ibuprofen, and cortisone) act in part by inhibiting their synthesis.  

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Wnt β-Catenin pathway

Wnt Proteins Bind to Frizzled Receptors and Inhibit the Degradation of β-Catenin They were discovered independently in flies and in mice: in Drosophila, the Wingless (Wg) gene originally came to light because of its role as a morphogen in wing development, while in mice, the Int1 gene was found because it promoted the development of breast tumors when activated by the integration of a virus next to it. Both of these genes encode Wnt proteins.There are 19 Wnts in humans, each having distinct, but often overlapping, functions.
 Wnts can activate at least two types of intracellular signaling pathway

1. Wnt/β-catenin pathway (also known as the canonical Wnt pathway), which is centered on the latent transcription regulator β-catenin.

2. Planar polarity pathway, coordinates the polarization of cells in the plane of a developing epithelium and depends on Rho family GTPases. Both of these pathways begin with the binding of Wnts to Frizzled.

The Wnt/β-catenin signaling pathway-

 (A) In the absence of a Wnt signal-

 β-catenin that is not bound to cell–cell adherens junctions interacts with a degradation complex containing APC, axin, GSK3, and CK1. In this complex, β-catenin is phosphorylated by CK1 and then by GSK3, triggering its ubiquitylation and degradation in proteasomes.

In the absence of Wnt signaling, Wnt-responsive genes are kept silent by an inhibitory complex of transcription regulatory proteins of the LEF1/TCF family bound to a co-repressor protein of the Groucho family . In response to a Wnt signal, β-catenin enters the nucleus and binds to the LEF1/TCF proteins, displacing Groucho. The β-catenin now functions as a coactivator, inducing the transcription of the Wnt target genes.Thus, Wnt/β-catenin signaling triggers a switch from transcriptional repression to transcriptional activation. Among the genes activated by β-catenin is Myc, which encodes a protein (Myc) that is an important regulator of cell growth and proliferation .

 (B) In the presence of wnt signal-

 Wnt proteins regulate β-catenin proteolysis by binding to both a Frizzled protein and a co-receptor that is related to the low-density lipoprotein (LDL) receptor  and is therefore called an LDL-receptor-related protein (LRP).  The activated receptor complex recruits the Dishevelled scaffold and the cytosolic tail of LRP is phosphorylated by  a protein kinase called casein kinase 1 (CK1) phosphorylates the β-catenin on a serine. For further phosphorylation by another protein kinase called glycogen synthase kinase 3 (GSK3); this final phosphorylation marks the protein for ubiquitylation and rapid degradation in proteasomes.

Two scaffold proteins called axin and Adenomatous polyposis coli (APC) hold the protein complex together .APC gets its name from the finding that the gene encoding it is often mutated in a type of benign tumor (adenoma) of the colon; the tumor projects into the lumen as a polyp and can eventually become malignant. (This APC should not be confused with the anaphase-promoting complex, or APC/C, that plays a central part in selective protein degradation during the cell cycle.

Axin binds to the phosphorylated LRP and is inactivated and/or degraded, resulting in disassembly of the degradation complex. The phosphorylation of β-catenin is thereby prevented, and unphosphorylated β-catenin accumulates and translocates to the nucleus, where it binds to LEF1/ TCF, displaces the co-repressor Groucho, and acts as a coactivator to stimulate the transcription of Wnt target genes. The scaffold protein Dishevelled is required for the signaling pathway to operate; it binds to Frizzled and becomes phosphorylated .

NOTE -The Wnt/β-catenin pathway acts by regulating the proteolysis of the multifunctional protein β-catenin (or Armadillo in flies).

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