Clinical Box 61 In Vivo Labeling of Cells

Cells monitor their environment through receptor proteins embedded in the cell membrane. Receptor proteins have several attached molecules of sugar or oligosaccharide chains protruding into the extracellular space. These oligosaccharide chains or glycans serve as binding points for ligands to the receptor protein. Developing the ability to detect glycans specific to a particular type of receptors or cells in the living patient would be a major advance in diagnosis and therapy.

A recent study (Prescher et al., 2004) has reported an important contribution toward this goal. Prescher and colleagues synthesized the precursor of a single sugar molecule or monosaccharide with an attached nontoxic azide group: peracetylated mannosamine. When injected into mice, this precursor was converted by the mouse cellular machinery into azide-bearing sialic acid and transferred to glycans in the cell membrane. Thus, membrane proteins on the mouse cells now contained this artificially manufactured azide-bearing sialic acid. When an appropriate probe, in this case phosphine attached to a peptide named Flag, was injected into these previously treated mice, it bound to the sialic acid. Cells were then harvested from the mice. The complex formed by the artificially induced azide-bearing sialic acid and phosphine attached to Flag was expressed on the cells as a tag, detectable by labeling with a fluorescent antibody specific to the Flag peptide.

This work has important implications because it is a first step in directing the cellular machinery to synthesize artificial tags in cells bearing a particular type of receptor. It has the potential to allow for the artificial labeling of a specific group of cells for detection or therapy in the living. An important step toward this goal would be to uncover the mechanism, which will direct the expression of the artificial tags only in desired tissues or cell types in the living patient. Future applications could focus on developing imaging methods for detecting the tagged cells in living patients for diagnostic purpose; for example, cells in a brain region could be tagged and checked for abnormal growth if a tumor is suspected. Furthermore, therapies can also be developed whereby antibodies specific for the artificial tags can seek out tag-labeled tumor cells and destroy them. This approach would be invaluable in areas difficult to access by surgery such as the brain.

monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) are responsible for transmitter termination. Although these enzymes play a major role in ending the effects of catecholamine in the bloodstream, they play a secondary role in the brain. The major method of transmitter termination in the brain is by way of catecholamine transporters. These specialized membrane proteins shuttle the catecholamines back into the presynaptic terminal in an energy-dependent process, which requires the presence of the salt ions sodium and chloride. These transporters differ from the vesicular transporters, which load neuro-transmitters into synaptic vesicles.

Two transporters specific to nerve cells have been characterized for the catecholamines: the dopamine transporter and the norepinephrine transporter. The existing nomenclature is somewhat misleading because the transporters are not specific and will bind to either dopamine or nor-epinephrine. The catecholamine transporters are located outside of the synapse; thus, following release and interaction with the receptors, the catecholamine neurotransmitters are terminated first passively by diffusion away from the synaptic cleft and then actively by their transporters. The arrangement of the transporters outside of the synaptic cleft allows for termination of catecholamines released not only from a nearby synapse, but also from distant sites. This mechanism increases the efficiency of communication between nerve cells by decreasing the likelihood of random neurotransmitter action through extracellular diffusion. Furthermore, it is protective to the cell since overstimulation can deplete metabolic stores within the postsynaptic cell and/or cause overexcitation within a particular neural pathway resulting in cell damage and death. This hypothesis of excitotoxicity may be the basis of severe neurological disorders such as strokes or amyotrophic lateral sclerosis.

6.3.2 Neuropeptide transmitters

Short chains of amino acids form neuropeptide transmitters. There is a large number of identified neuropeptide transmitters with new ones discovered on a regular basis. Substance P, vasoactive intestinal peptide, cholecystokinin, somatostatin, and neurotensin are examples of neu-ropeptide transmitters acting not only in the nervous system but also in the gastrointestinal tract as paracrines and hormones (see Sec. 6.4).

Although they play a similar role in cell communication, neuropep-tide transmitters differ from the classical transmitters in some aspects. Synthesis of neuropeptides occurs in the cell body or soma of nerve cells, far away from their site of release. Cell transport mechanisms shuttle the neuropeptides from the cell body to the axonal terminals. Given the constraint of longer transport of the peptide neurotransmit-ter to the axon terminal and slower transmitter replenishment, the release of the neuropeptide transmitter must be coordinated with its synthesis to avoid shortage.

Although storage of the neuropeptide transmitters takes place in vesicles that are twice as large as the vesicles observed with the classical neurotransmitters, synaptic mechanisms involved in the release of the neuropeptide transmitters appear to be similar to those involving classical transmitters.

Finally, the action of neuropeptide transmitters is terminated passively by diffusion away from the synapse or actively by enzyme degradation. Thus far, no reuptake process via transporters has been identified for the neuropeptide transmitters.

6.4 Cell Secretion

Cells communicate with themselves and with each other by multiple means. Chemical communication can occur in an endocrine (humoral), paracrine, autocrine, or juxtacrine way. These terms were already introduced in Chap. 4 and they are again briefly explained below. In order to communicate chemically, the cell must synthesize a substance intra-cellularly and release it extracellularly. This process is called secretion which is the broad topic of this section.

Endocrine: The endocrine system is characterized by humoral communication in which a cell synthesizes a hormone and secretes it directly into the circulatory system. The blood transports the hormone to a target tissue where it binds to receptors located on the membrane or inside cells to promote its effects. Application Box 6.1 presents bioengineering attempts to restore in diabetic patients communication between cells that produce the hormone insulin and cells that express insulin receptors.

Paracrine: In paracrine communication, cells release active substances, which affect nearby cells.

Autocrine: A cell releases a substance, which acts upon the same cell. Juxtacrine: In this type of communication, the messenger acts on neighboring cells without diffusing from the cell where it was produced. For instance, a ligand anchored in the cell membrane binds to a receptor in the membrane of another cell.

APPLICATION BOX 6.1 The Artificial Pancreas of the Future

All cells use glucose to derive energy for their functions. The hormone insulin allows for the passage of glucose from the bloodstream into cells. Without insulin, glucose builds up in the bloodstream and is unavailable for cell use.

In patients with type 1 diabetes (juvenile diabetes), b cells from the pancreas stop secreting insulin. At present, one possible therapy is transplantation of pancreatic tissues from cadavers to restore insulin secretion. However, the disadvantages of this method include having to use two separate pancreas donors for each recipient because only a fraction of the transplanted tissue will become functional; further, the rate of independence from injected insulin in these patients decreases over time: from 80% at 1 year to 65% by 2 years. A powerful potential therapy eliminating the need for donors would be the use of artificial b cells.

Bioengineering has contributed enormously to the development of improved treatments of diabetes. Implantable insulin pumps have been continuously improved since their first introduction in the early eighties. The pumps are nowadays connected with a miniature glucose sensor, and at the current writing, the first real-time monitoring systems have just been approved for use in humans. It implies that for diabetics, the widespread use of implanted closed-loop insulin delivery systems that mimic the functions of the human pancreas may not be in the too distant future.

There are other experimental approaches to the treatment of diabetes in which bioengineering design may be important. For instance, bioengi-neers aim at developing an implantable membrane device that entraps b cells isolated from animals or human donors and grown in culture. The membrane has to be designed to be freely permeable to glucose and insulin. But the membrane must also protect the entrapped b cells from immune factors such as antibodies and cytokines from the implanted patient, since they can elicit an immune reaction against the implanted device and interfere with its function. Further research is needed to develop such a valuable device.

Though most attention has been focused on chemical transmission as the generally accepted mode of cell to cell communication, electrical communication exists as well, in the form of gap junctions and ephaptic information exchange.

Gap junctions: Communication occurs through protein channels that form in the apposed membranes of adjacent cells and allow the exchange of small molecules and ions between the cells. The flow of ions allows electric impulses to be transmitted directly from one cell to the other.

Ephaptic: Cells communicate by apposed cell membranes with regions of low resistance flanked by regions of high resistance. Electrical signals flow across membranes at regions of low resistance.

Cellular secretion is a complex and highly regulated task. It first involves manufacturing of the substance to be secreted (see Sec. 6.4.1). The secretory cell has to synthesize two types of products: those destined for internal use and those destined for secretion, that is, release to the external environment. Next, the synthesized products must be packaged for efficient transport to their destination and also to prevent them from acting prematurely. These processes are presented under packaging in Sec. 6.4.2. The packages must have specific labels because they will be delivered by different systems to their respective destinations. The processes involved in sorting and delivery are crucial aspects of secretion and explained in Sec. 6.4.3. Finally, the products destined for secretion can only be secreted when there is a need for them and therefore secretion is highly regulated as presented in Sec. 6.4.4. The fusion of the secretory vesicle with the cell membrane and the release of their content extracellularly is called exocytosis, and presented in Sec. 6.4.5.

6.4.1 Manufacturing

Peptides or proteins formed on free ribosomes are usually destined for intracellular processes. Proteins destined for secretion are manufactured in ribosomes bound to the endoplasmic reticulum. The latter is called rough endoplasmic reticulum (RER) because of its high content of ribosomes and its appearance in electron micrographs. RER can be seen in Chap. 2 in the schematic of Fig. 2.1 and in the electron micrograph of Fig. 2.5. The process of synthesizing peptides and proteins from RNAis called translation and is described in Sec. 7.5.

6.4.2 Packaging

As proteins leave the RER, they are shuttled to the Golgi apparatus by way of the endoplasmic reticulum Golgi intermediate compartment

Figure 6.13 Endoplasmic reticulum Golgi intermediate compartment (ERGIC). The ERGIC, also called vesicular tubular cluster (VTC), shuttles synthesized proteins from the endoplasmic reticulum (ER) to the Golgi apparatus for processing and packaging. (Reproduced, with permission from the Annual Review of Cell and Developmental Biology 20, 2004, by Annual Reviews http://www. annualreviews.org.)

Figure 6.13 Endoplasmic reticulum Golgi intermediate compartment (ERGIC). The ERGIC, also called vesicular tubular cluster (VTC), shuttles synthesized proteins from the endoplasmic reticulum (ER) to the Golgi apparatus for processing and packaging. (Reproduced, with permission from the Annual Review of Cell and Developmental Biology 20, 2004, by Annual Reviews http://www. annualreviews.org.)

(ERGIC), also called vesicular tubular cluster (VTC) (Fig. 6.13). During this process, the secretory proteins are wrapped in transport vesicles which bud from the RER exit sites. These transport vesicles, known as COPII-coated vesicles, will move by way of interconnected tubules in the ERGIC or along microtubules to the cis-face of the Golgi apparatus.

The Golgi complex is organized into the cis-Golgi network (entry site) and the trans-Golgi network (exit site), which is shown in Fig. 2.6 as an electron micrograph and in schematic form. The cis and trans sites are both sorting stations: the cis site separating proteins destined for return to the RER from those continuing into the Golgi complex, and the trans site triaging proteins destined for the cell membrane from those with intra-cellular destinations. Within the Golgi complex, proteins are further modified by glycosylation, that is, attachment of carbohydrate entities, as they are shuttled toward the trans-Golgi network, the exit point of the Golgi complex. In case vital proteins are needed again in the RER, the Golgi complex has a mechanism for trapping them and sending them back to the RER via retrograde transport in so-called COPI vesicles (Fig. 6.13).

At the Golgi exit site, the secretory proteins will be packaged, initially into pieces of membrane from the trans-Golgi network, which loosely wrap themselves around the aggregates of synthesized secretory proteins. The resulting structures are called immature secretory vesicles. The signals directing the proteins into aggregates and the signals packaging the aggregates into secretory vesicles remain unknown. However, they may be common to all secretory cells because cells that normally do not synthesize a particular protein will still package that protein appropriately if forced to express the gene for that protein by artificial methods.

A number of processes take place further in the interior of the secretory vesicles as they mature. The vesicles concentrate their content by continuous removal of portions of their membrane, which are then recycled back to late endosomes or to the Golgi apparatus. A progressive acidification of the interior of the secretory vesicles also occurs due to an increase in ATP-dependent H+ pumps in the vesicle membrane. Finally, the protein content of the secretory vesicles may be processed from an inactive precursor form to an active form by cleavage. For example, the secretory vesicle may contain a single precursor polyprotein, which will be cleaved off into multiple end-products. All these steps result in a more efficient packaging system for delivery.

6.4.3 Sorting and delivery

Secretory vesicles once formed must move toward their target membrane for eventual fusion and release of their content. It remains unclear as to how cells label the secretory vesicles for specific destinations. The delivery is performed by a system of microtubules, which shuttle the vesicles, and once they arrive, the vesicles become tethered to their target membrane, mainly by a group of GTP-binding proteins named Rabs. The specificity of the secretory vesicles for their target membrane is determined by the type of Rabs. It is believed that this tethering process is the earliest step in the fusion of the secretory vesicle with its target membrane.

The process by which the secretory vesicle interacts with the target membrane is known as docking, and it is performed by the interaction of two different categories of proteins known as SNAREs. The latter are formed by the strong binding of three synaptic proteins shown in Fig. 6.14: the vesicle-associated membrane protein (VAMP) or synaptobrevin, syntaxin, and the synaptosomal-associated protein of 25 kDa (SNAP-25). The binding of these three synaptic proteins form the SNAP receptor complex also known as SNARE. SNAREs confer further specificity to the secretory vesicle for the fusion process to its target membrane. Secretory vesicles contain v-SNAREs, incorporated into their membrane during their budding from the trans-Golgi network. Target membranes contain t-SNAREs, interacting to form complexes with the approaching v-SNAREs, pulling the lipid bilayers of the secretory vesicle and target membrane into contact for fusion.

Figure 6.14 Docking mechanism of the synaptic vesicle to the presynaptic membrane. The docking mechanism involves binding of synaptobrevin, syntaxin, and SNAP-25, which form the SNARE complex.

6.4.4 Regulation of secretion

Since secretion occurs only on demand, upon reception of specific signals, interaction of the SNAREs is by itself not sufficient for membrane fusion. For example, in nerve cells, the v- and t-SNARE complexes remain in an inactive conformation due to the binding of complexin, and the secretory vesicle remains docked with its target membrane, awaiting the activating signal for fusion. This triggering signal in most cells will be an increase in calcium ion concentration.

Once fusion occurs, the SNAREs will be removed by the binding of a protein named A-ethylmaleimide sensitive factor (NSF) or A-ethylmaleimide sensitive fusion protein. NSF first binds a cofactor called a-soluble NSF attachment protein (a-SNAP), and this complex then binds to the SNARE. The latter will be reduced to its component proteins in an energy-dependent process by hydrolysis of adenosine triphosphate (ATP) and returned to its dissociated state.

6.4.5 Exocytosis

Exocytosis is a common process of many cells, but has been studied best at synapses, the contact points between nerve cells or between nerve cells and their target tissues. As an electric impulse arrives at the nerve terminal, an influx of calcium ions is triggered and the SNARE complex pulls the lipid bilayers of the secretory vesicle and its target membrane together (Fig. 6.14). In nerve cells, this fusion is regulated by synapto-tagmin, a calcium-binding protein. It has been shown by in vitro experiments that the pulling action of SNAREs is enough to trigger fusion of the membranes. During this process, the cytosolic face of the secretory vesicle (that portion of the membrane of the secretory vesicle which is turned toward the outside of the vesicle) becomes part of the cytoso-lic surface of the cell membrane. The luminal surface of the vesicle

(that is the portion of the membrane of the secretory vesicle turned toward the inside of the vesicle) becomes part of the outer surface of the cell membrane. The end result is the formation of a "fusion pore" illustrated in Fig. 6.10. The fusion pore rapidly dilates to release the content of the secretory vesicle.

Interactions between READ and WRITE of the Signaling Machinery

6.5 Synaptic Interactions during Development

Much is known about the molecular interactions between pre- and postsynaptic structures, the READ and WRITE mechanisms in our model of signaling machinery. A large part of this knowledge is acquired from studies that investigate the synaptic interactions during development, specifically of the neuromuscular junction, the interface between a voluntary nerve fiber and a muscle cell (Kummer et al., 2006). The voluntary nerve fibers are derived from motoneurons in the spinal cord and their terminals will interact with cholinergic receptors of the nicotinic types (see Sec. 6.3) on the muscle membrane.

During development, a pre-pattern of dense clusters of nicotinic cholinergic receptors forms on the muscle cell membrane. These clusters are expressed well in advance of the arrival of the motoneuron axon to the muscle cell. It was originally assumed that the presence of the motoneuron axon is necessary for expression of receptor clusters on the muscle cell membrane. But recent results have shown that the muscle cell can express this pre-pattern of nicotinic cholinergic receptors on its own. In fact, tissue culture studies have shown that muscle cells can form complex clusters of cholinergic receptors, which resemble neuromuscular junctions. These complex clusters, named "aneural pretzels" are shaped under the influence of a muscle-derived protein named LL5j6. Thus, the shape of the mature neuromuscular junction is the result of interactions between muscle-derived and nerve-derived factors.

As the axon terminals from the motoneuron contact the muscle cell membrane, some of the pre-pattern clusters disappear whereas others are incorporated into the neuromuscular junction. The pre-pattern receptor clusters appear to play an important role in attracting and stabilizing the incoming axon terminals. Once neuronal contact has been established, the presence of the axon terminals is required for continued existence of the neuromuscular junction: if the axon terminals do not make contact, the nicotinic cholinergic receptor clusters eventually disappear.

Interestingly, the presence of the neurotransmitter acetylcholine (ACh) promotes the loss of the pre-pattern clusters of nicotinic cholinergic receptors and the incoming axon terminals secrete agrin, to counteract this effect. Agrin, a large heparan sulfate proteoglycan, stabilizes the pre-pattern receptor clusters contacted by axon terminals, emphasizing the importance of the presynaptic influence on the receptor distribution pattern in the postsynaptic element.

Additionally, the presynaptic nerve fiber also influences elements other than the postsynaptic structure: presynaptic axon terminals secrete neuregulin, which influences surrounding nonneuronal cells called Schwann cells. These play an important supportive role in maintaining the neuromuscular junction.

Finally, the type of neurotransmitter synthesized by the presynaptic element strongly influences the type of receptors manufactured by the postsynaptic structure. Recent results (Spitzer et al., 2004) have shown that the level of calcium activity inside a nerve cell during development can influence the type of neurotransmitters produced: an increase in calcium activity results in a shift to the production of the inhibitory transmitter g-aminobutyric acid (GABA), whereas a decrease leads to synthesis of the excitatory transmitter glutamate. Surprisingly, when glutamatergic axon terminals contact muscle cells, which normally express cholinergic receptors, the muscle cells respond by manufacturing glutamate receptors, demonstrating not only the influence of the presy-naptic element on the postsynaptic structure, but also the versatility of the postsynaptic element in adapting to new or different stimuli.

In summary, the study of molecular interactions between pre- and postsynaptic elements during development allows for a glimpse of the complex interplay necessary in sculpting and maintaining the communication between cells. It also points to potential approaches in manipulating or designing agents, which can shape or repair synaptic formation, an invaluable therapeutic step in developmental or degenerative disorders.

References

Kummer, T.T., Misgeld, T. and Sanes, J.R. (2006). Assembly of the postsynaptic membrane at the neuromuscular junction: Paradigm lost. Curr. Opin. Neurobiol. 16:74-82. Nishi, M. and Kawata, M. (2006). Brain corticosteroid receptor dynamics and trafficking:

Implications from live cell imaging. Neuroscientist. 12:119-133. Prescher, J.A., Dube, D.H. and Bertozzi CR (2004). Chemical remodelling of cell surfaces in living animals. Nature. 430:873-877. Spitzer, N.C., Root, C.M. and Borodinsky, L.N. (2004). Orchestrating neuronal differentiation: Patterns of Ca2+ spikes specify transmitter choice. Trends Neurosci. 27:415-421.

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