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Summary of Cell Structure, Anatomic Correlates of Metabolic Function

Author and Curator: Larry H. Bernstein, MD, FCAP  

 

This chapter has been concerned with the subcellular ultrastructure of organelles, and importantly, their function.  There is no waste in the cell structure. The nucleus has the instructions necessary to carry out the cell’s functions.  In the Eukaryotic cell there is significant differentiation so that the cells are regulated for the needs that they uniquely carry out.  When there is disregulation, it leads to remodeling or to cell death.

Here I shall note some highlights of this chapter.

1. In every aspect of cell function, proteins are involved embedded in the structure, for most efficient functioning.
2. Metabolic regulation is dependent on pathways that are also linkages of proteins.
3. Energy utilization is dependent on enzymatic reactions, often involving essential metal ions of high valence numbers, which facilitates covalent and anion binding, and has an essential role in allostericity.

Mitochondria

http://en.wikipedia.org/wiki/File:Mitochondria,_mammalian_lung_-_TEM.jpg

Mitochondria range from 0.5 to 1.0 micrometer (μm) in diameter. These structures are sometimes described as “cellular power plants” because they generate most of the cell’s supply of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in other tasks such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunction.

The number of mitochondria in a cell can vary widely by organism, tissue, and cell type. For instance, red blood cells have no mitochondria, whereas liver cells can have more than 2000. The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. The mitochondrial proteome is thought to be dynamically regulated. Although most of a cell’s DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes.

In 1913 particles from extracts of guinea-pig liver were linked to respiration by Otto Heinrich Warburg, which he called “grana”. Warburg and Heinrich Otto Wieland, who had also postulated a similar particle mechanism, disagreed on the chemical nature of the respiration. It was not until 1925 when David Keilin discovered cytochromes that the respiratory chain was described.  In 1939, experiments using minced muscle cells demonstrated that one oxygen atom can form two adenosine triphosphate molecules, and, in 1941, the concept of phosphate bonds being a form of energy in cellular metabolism was developed by Fritz Albert Lipmann. In the following years, the mechanism behind cellular respiration was further elaborated, although its link to the mitochondria was not known. The introduction of tissue fractionation by Albert Claude allowed mitochondria to be isolated from other cell fractions and biochemical analysis to be conducted on them alone. In 1946, he concluded that cytochrome oxidase and other enzymes responsible for the respiratory chain were isolated to the mitchondria.

The first high-resolution micrographs appeared in 1952, replacing the Janus Green stains as the preferred way of visualising the mitochondria. This led to a more detailed analysis of the structure of the mitochondria, including confirmation that they were surrounded by a membrane. It also showed a second membrane inside the mitochondria that folded up in ridges dividing up the inner chamber and that the size and shape of the mitochondria varied from cell to cell.  In 1967, it was discovered that mitochondria contained ribosomes. In 1968, methods were developed for mapping the mitochondrial genes, with the genetic and physical map of yeast mitochondria being completed in 1976.

A mitochondrion contains outer and inner membranes composed of phospholipid bilayers and proteins. The two membranes have different properties. Because of this double-membraned organization, there are five distinct parts to a mitochondrion. They are:

1. the outer mitochondrial membrane,
2. the intermembrane space (the space between the outer and inner membranes),
3. the inner mitochondrial membrane,
4. the cristae space (formed by infoldings of the inner membrane), and
5. the matrix (space within the inner membrane).

Mitochondria stripped of their outer membrane are called mitoplasts.

http://upload.wikimedia.org/wikipedia/commons/thumb/9/9e/Mitochondrion_structure_drawing.svg/500px-Mitochondrion_structure_drawing.svg.png

Mitochondrion ultrastructure (interactive diagram) A mitochondrion has a double membrane; the inner one contains its chemiosmotic apparatus and has deep grooves which increase its surface area. While commonly depicted as an “orange sausage with a blob inside of it” (like it is here), mitochondria can take many shapes and their intermembrane space is quite thin.

The intermembrane space is the space between the outer membrane and the inner membrane. It is also known as perimitochondrial space. Because the outer membrane is freely permeable to small molecules, the concentrations of small molecules such as ions and sugars in the intermembrane space is the same as the cytosol. However, large proteins must have a specific signaling sequence to be transported across the outer membrane, so the protein composition of this space is different from the protein composition of the cytosol. One protein that is localized to the intermembrane space in this way is cytochrome c.

The inner mitochondrial membrane contains proteins with five types of functions:

1. Those that perform the redox reactions of oxidative phosphorylation
2. ATP synthase, which generates ATP in the matrix
3. Specific transport proteins that regulate metabolite passage into and out of the matrix
4. Protein import machinery.
5. Mitochondria fusion and fission protein.

It contains more than 151 different polypeptides, and has a very high protein-to-phospholipid ratio (more than 3:1 by weight, which is about 1 protein for 15 phospholipids). The inner membrane is home to around 1/5 of the total protein in a mitochondrion. In addition, the inner membrane is rich in an unusual phospholipid, cardiolipin. This phospholipid was originally discovered in cow hearts in 1942, and is usually characteristic of mitochondrial and bacterial plasma membranes. Cardiolipin contains four fatty acids rather than two, and may help to make the inner membrane impermeable. Unlike the outer membrane, the inner membrane doesn’t contain porins, and is highly impermeable to all molecules. Almost all ions and molecules require special membrane transporters to enter or exit the matrix. Proteins are ferried into the matrix via the translocase of the inner membrane (TIM) complex or via Oxa1. In addition, there is a membrane potential across the inner membrane, formed by the action of the enzymes of the electron transport chain.

The inner mitochondrial membrane is compartmentalized into numerous cristae, which expand the surface area of the inner mitochondrial membrane, enhancing its ability to produce ATP. For typical liver mitochondria, the area of the inner membrane is about five times as large as the outer membrane. This ratio is variable and mitochondria from cells that have a greater demand for ATP, such as muscle cells, contain even more cristae. These folds are studded with small round bodies known as F₁ particles or oxysomes. These are not simple random folds but rather invaginations of the inner membrane, which can affect overall chemiosmotic function. One recent mathematical modeling study has suggested that the optical properties of the cristae in filamentous mitochondria may affect the generation and propagation of light within the tissue.

http://upload.wikimedia.org/wikipedia/commons/thumb/d/d8/MitochondrionCAM.jpg/250px-MitochondrionCAM.jpg

The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the total protein in a mitochondrion. The matrix is important in thThe MAM is enriched in enzymes involved in lipid biosynthesis, such as phosphatidylserine synthase on the ER face and phosphatidylserine decarboxylase on the mitochondrial face.^[28][29] Because mitochondria are dynamic organelles constantly undergoing fission and fusion events, they require a constant and well-regulated supply of phospholipids for membrane integrity.^[30][31] But mitochondria are not only a destination for the phospholipids they finish synthesis of; rather, this organelle also plays a role in inter-organelle trafficking of the intermediates and products of phospholipid biosynthetic pathways, ceramide and cholesterol metabolism, and glycosphingolipid anabolisme production of ATP with the aid of the ATP synthase contained in the inner membrane. The matrix contains a highly concentrated mixture of hundreds of enzymes, special mitochondrial ribosomes, tRNA, and several copies of the mitochondrial DNA genome. Of the enzymes, the major functions include oxidation of pyruvate and fatty acids, and the citric acid cycle.

Purified MAM from subcellular fractionation has shown to be enriched in enzymes involved in phospholipid exchange, in addition to channels associated with Ca²⁺ signaling. The mitochondria-associated ER membrane (MAM) is another structural element that is increasingly recognized for its critical role in cellular physiology and homeostasis. Once considered a technical snag in cell fractionation techniques, the alleged ER vesicle contaminants that invariably appeared in the mitochondrial fraction have been re-identified as membranous structures derived from the MAM—the interface between mitochondria and the ER. Physical coupling between these two organelles had previously been observed in electron micrographs and has more recently been probed with fluorescence microscopy. Such studies estimate that at the MAM, which may comprise up to 20% of the mitochondrial outer membrane, the ER and mitochondria are separated by a mere 10–25 nm and held together by protein tethering complexes.

Such trafficking capacity depends on the MAM, which has been shown to facilitate transfer of lipid intermediates between organelles. In contrast to the standard vesicular mechanism of lipid transfer, evidence indicates that the physical proximity of the ER and mitochondrial membranes at the MAM allows for lipid flipping between opposed bilayers. Despite this unusual and seemingly energetically unfavorable mechanism, such transport does not require ATP. Instead, in yeast, it has been shown to be dependent on a multiprotein tethering structure termed the ER-mitochondria encounter structure, or ERMES, although it remains unclear whether this structure directly mediates lipid transfer or is required to keep the membranes in sufficiently close proximity to lower the energy barrier for lipid flipping.

A critical role for the ER in calcium signaling was acknowledged before such a role for the mitochondria was widely accepted, in part because the low affinity of Ca²⁺ channels localized to the outer mitochondrial membrane seemed to fly in the face of this organelle’s purported responsiveness to changes in intracellular Ca²⁺ flux. But the presence of the MAM resolves this apparent contradiction: the close physical association between the two organelles results in Ca²⁺ microdomains at contact points that facilitate efficient Ca²⁺ transmission from the ER to the mitochondria. Transmission occurs in response to so-called “Ca²⁺ puffs” generated by spontaneous clustering and activation of IP3R, a canonical ER membrane Ca²⁺ channel.

The properties of the Ca²⁺ pump SERCA and the channel IP3R present on the ER membrane facilitate feedback regulation coordinated by MAM function. In particular, clearance of Ca²⁺ by the MAM allows for spatio-temporal patterning of Ca²⁺ signaling because Ca²⁺ alters IP3R activity in a biphasic manner. SERCA is likewise affected by mitochondrial feedback: uptake of Ca²⁺ by the MAM stimulates ATP production, thus providing energy that enables SERCA to reload the ER with Ca²⁺ for continued Ca²⁺ efflux at the MAM. Thus, the MAM is not a passive buffer for Ca²⁺ puffs; rather it helps modulate further Ca²⁺ signaling through feedback loops that affect ER dynamics.

Regulating ER release of Ca²⁺ at the MAM is especially critical because only a certain window of Ca²⁺ uptake sustains the mitochondria, and consequently the cell, at homeostasis. Sufficient intraorganelle Ca²⁺ signaling is required to stimulate metabolism by activating dehydrogenase enzymes critical to flux through the citric acid cycle. However, once Ca²⁺ signaling in the mitochondria passes a certain threshold, it stimulates the intrinsic pathway of apoptosis in part by collapsing the mitochondrial membrane potential required for metabolism.  Studies examining the role of pro- and anti-apoptotic factors support this model; for example, the anti-apoptotic factor Bcl-2 has been shown to interact with IP3Rs to reduce Ca²⁺ filling of the ER, leading to reduced efflux at the MAM and preventing collapse of the mitochondrial membrane potential post-apoptotic stimuli. Given the need for such fine regulation of Ca²⁺ signaling, it is perhaps unsurprising that dysregulated mitochondrial Ca²⁺ has been implicated in several neurodegenerative diseases, while the catalogue of tumor suppressors includes a few that are enriched at the MAM.

…more

http://en.wikipedia.org/wiki/Mitochondrion

Lysosome and Apoptosis

Role of autophagy in cancer

R Mathew, V Karantza-Wadsworth & E White

Nature Reviews Cancer 7, 961-967 (Dec 2007) |  http://dx.doi.org:/10.1038/nrc2254

Autophagy is a cellular degradation pathway for the clearance of damaged or superfluous proteins and organelles. The recycling of these intracellular constituents also serves as an alternative energy source during periods of metabolic stress to maintain homeostasis and viability. In tumour cells with defects in apoptosis, autophagy allows prolonged survival. Paradoxically, autophagy defects are associated with increased tumorigenesis, but the mechanism behind this has not been determined. Recent evidence suggests that autophagy provides a protective function to limit tumour necrosis and inflammation, and to mitigate genome damage in tumour cells in response to metabolic stress.

Sustained Activation of mTORC1 in Skeletal Muscle Inhibits Constitutive and Starvation-Induced Autophagy and Causes a Severe, Late-Onset Myopathy

P Castets, S Lin, N Rion, S Di Fulvio, et al.
cell-metabolism 7 May, 2013; 17(5): p731–744   http://dx.doi.org/10.1016/j.cmet.2013.03.015

• mTORC1 inhibition is required for constitutive and starvation-induced autophagy
• Sustained activation of mTORC1 causes a severe myopathy due to autophagy impairment
• TSC1 depletion is sufficient to activate mTORC1 irrespective of other stimuli
• mTORC1 inactivation is sufficient to trigger LC3 lipidation

Autophagy is a catabolic process that ensures homeostatic cell clearance and is deregulated in a growing number of myopathological conditions. Although FoxO3 was shown to promote the expression of autophagy-related genes in skeletal muscle, the mechanisms triggering autophagy are unclear. We show that TSC1-deficient mice (TSCmKO), characterized by sustained activation of mTORC1, develop a late-onset myopathy related to impaired autophagy. In young TSCmKO mice,

• constitutive and starvation-induced autophagy is blocked at the induction steps via
• mTORC1-mediated inhibition of Ulk1, despite FoxO3 activation.

Rapamycin is sufficient to restore autophagy in TSCmKO mice and

• improves the muscle phenotype of old mutant mice.

Inversely, abrogation of mTORC1 signaling by

• depletion of raptor induces autophagy regardless of FoxO inhibition.

Thus, mTORC1 is the dominant regulator of autophagy induction in skeletal muscle and

• ensures a tight coordination of metabolic pathways.

These findings may open interesting avenues for therapeutic strategies directed toward autophagy-related muscle diseases.

Histone deacetylases 1 and 2 regulate autophagy flux and skeletal muscle homeostasis in mice

Viviana Moresi, et al.   PNAS Jan 31, 2012; 109(5): 1649-1654
http://dx.doi.org:/10.1073/pnas.1121159109
http://www.pnas.org/content/109/5/1649/F6.medium.gif

HDAC1 activates FoxO and is both sufficient and required for skeletal muscle atrophy

Beharry, PB. Sandesara, BM. Roberts, et al.
J. Cell Sci. Apr 2014 127 (7) 1441-1453   http://dx.doi.org:/10.1242/​jcs.136390

The Forkhead box O (FoxO) transcription factors are activated, and necessary for the muscle atrophy, in several pathophysiological conditions, including muscle disuse and cancer cachexia. However, the mechanisms that lead to FoxO activation are not well defined. Recent data from our laboratory and others indicate that

• the activity of FoxO is repressed under basal conditions via reversible lysine acetylation,
• which becomes compromised during catabolic conditions.

Therefore, we aimed to determine how histone deacetylase (HDAC) proteins contribute to

• activation of FoxO and induction of the muscle atrophy program.

Through the use of various pharmacological inhibitors to block HDAC activity, we demonstrate that

• class I HDACs are key regulators of FoxO and the muscle-atrophy program
• during both nutrient deprivation and skeletal muscle disuse.

Furthermore, we demonstrate, through the use of wild-type and dominant-negative HDAC1 expression plasmids,

• that HDAC1 is sufficient to activate FoxO and induce muscle fiber atrophy in vivo and
• is necessary for the atrophy of muscle fibers that is associated with muscle disuse.

The ability of HDAC1 to cause muscle atrophy required its deacetylase activity and

• was linked to the induction of several atrophy genes by HDAC1,
• including atrogin-1, which required deacetylation of FoxO3a.

Moreover, pharmacological inhibition of class I HDACs during muscle disuse, using MS-275,

• significantly attenuated both disuse muscle fiber atrophy and contractile dysfunction.

Together, these data solidify the importance of class I HDACs in the muscle atrophy program and

• indicate that class I HDAC inhibitors are feasible countermeasures to impede muscle atrophy and weakness.

Autophagy and thyroid carcinogenesis: genetic and epigenetic links
F Morani, R Titone, L Pagano, et al.  Endocr Relat Cancer Feb 1, 2014 21 R13-R29
http://dx.doi.org:/10.1530/ERC-13-0271

Autophagy is a vesicular process for the lysosomal degradation of protein aggregates and

• of damaged or redundant organelles.

Autophagy plays an important role in cell homeostasis, and there is evidence that

• this process is dysregulated in cancer cells.

Recent in vitro preclinical studies have indicated that autophagy is

• involved in the cytotoxic response to chemotherapeutics in thyroid cancer cells.

Indeed, several oncogenes and oncosuppressor genes implicated in thyroid carcinogenesis

• also play a role in the regulation of autophagy.

In addition, some epigenetic modulators involved in thyroid carcinogenesis also influence autophagy. In this review, we highlight the genetic and epigenetic factors that

• mechanistically link thyroid carcinogenesis and autophagy, thus substantiating the rationale for
• an autophagy-targeted therapy of aggressive and radio-chemo-resistant thyroid cancers.