What system of internal membranes are found in eukaryotic cells?

Alternative Splicing

Eukaryotic cells have applied the mechanics of RNA splicing to generate the protein diversity necessary to meet their multiple demands. Thus, in contrast with the original definition of a gene in which only one transcript is produced, complex genes can generate multiple protein isoforms from multiple RNA transcripts through alternative splicing (433). This can be achieved by altering which introns and exons are included in or excluded from the mature mRNA transcript that is used as the template for peptide chain elongation. Accordingly, the definition of introns and exons for each gene is actually a fluid concept because an intron for one gene product may become an exon within another transcript. Alternative splicing is a mechanism used by many protein classes, including muscle-related genes, hormones, and transcription factors (434–438).

Abstract

Eukaryotic cells present an intricate network of intracellular membranes, which defines the nucleus and other organelles with distinct biochemical composition, structure, and functions. Additional cell components, such as the cytoskeleton, ribosome, proteasome and centrosome, are unbound of membrane. Consequently, subcellular compartmentalization of membrane-bound or membrane-unbound organelles has allowed for spatial control of biological processes. The differential subcellular distribution of molecules has also permitted to evolve distinct temporal regulations of individual biological processes. In this chapter, we will introduce basic aspects to understand the cell function having the major organization of the mammalian epithelial cell organelles as a reference. Along the text, genetic cases presenting fundamental clinical and/or laboratorial findings of a monogenic disease leading to specific organelle dysfunction will illustrate some functional roles discussed in each section.

Membrane structure

Eukaryotic cells, like prokaryotic cells, are surrounded by a plasma membrane. However, most eukaryotic cells also contain extensive internal membranes that enclose specific compartments (organelles) and separate them from the cytoplasm. Most organelles are surrounded by a single phospholipid bilayer membrane, but several, including the nucleus and the mitochondria, are enclosed by two membranes. These membranes control the ionic composition of organelles and spatially segregate metabolic pathways within the cell. The plasma membrane is a thin (5 nm) bilayer of lipids and proteins. Plasma membrane lipids are chemically diverse, but phospholipids are the most abundant. Phospholipid molecules are amphipathic and spontaneously form bilayers in water. They typically contain either a glycerol (as in phosphoglycerides) or a sphingosine (as in sphingomyelin) backbone. The plasma membrane also contains cholesterol, which confers rigidity. Under physiologic conditions, lipids and integral membrane proteins can diffuse laterally through the membrane leaflet (they do not usually migrate from one leaflet of a bilayer to the other), a concept known as the fluid mosaic model. The two sides (faces) of the lipid bilayer have different lipid compositions. Inositol phospholipids, which are substrates for enzymes that create second messengers such as inositol trisphosphate (Chapter 3), tend to be concentrated on the cytoplasmic face. Glycolipids (i.e. lipids with sugars attached to their head groups) are found on the extracellular face. Glycolipids are prominent in the myelin membrane that sheaths axons.

Pure lipid bilayers allow hydrophobic and small uncharged polar molecules to pass through, but largely block the diffusion of ions and larger polar molecules. Biologic membranes contain two major types of membrane transport protein to facilitate selective permeability to ions and large molecules. Carrier proteins have at least two different conformational states, which bind a solute on one side of the membrane and release it on the other. Channel proteins span the membrane and allow the passage of molecules across the membrane. Channel proteins allow more rapid transfer than carrier proteins, but only facilitate the diffusion of certain molecules down electrochemical gradients. Most channel proteins are highly selective for particular molecules or ions. In general, channels are not constantly open, but act as gated pores and open in response to a particular stimulus. Carrier proteins, by comparison, can actively transport compounds; they are often pumps driven by the energy derived from ATP hydrolysis or by the energy stored in ion gradients.

Plasma Membrane

Eukaryotic cells are encapsulated by a plasma membrane that makes the cell selectively permeable to many extracellular factors, including nutrients, lipids, proteins, ions, and pathogens. The plasma membrane is a fluid lipid bilayer containing a complex mixture of phospholipids, glycolipids, sterols, and proteins. This structure serves as an effective barrier to molecules that are poorly lipid soluble and also plays a critical role in the bidirectional flow of information. This conduction of information includes the specific recognition of extracellular factors, including hormones, toxins, adhesion molecules, and pathogens, that function to modulate cellular responses. As previously noted, the recognition of these factors at the plasma membrane is mediated by receptors, and receptor engagement elicits changes in cell behavior through the alteration of intracellular processes (signal transduction). These processes entail changes in the action of various enzymes, structural proteins, adapter molecules, and transcription factors. In addition, the regulated flow of ions (e.g., Ca2+) across the plasma membrane can modulate various signaling events.

Another important feature of the plasma membrane is the presence of specialized microdomains (“lipid rafts”) that consist of a unique composition of sterols, lipids, and proteins. These localized differences in membrane structure promote the recruitment of certain receptors and associated molecules to these regions, facilitating the rapid activation of these signaling complexes in response to appropriate stimuli.

Extracellular Matrix

All eukaryotic cells are surrounded by a characteristic extracellular matrix (ECM) composed of small molecules, collagens, elastin, and other proteoglycans and glycoproteins [111,112]. ECM regulates normal tissue homeostasis. In disease, healthy tissue is disrupted resulting in ECM remodeling during disease progression [112–114]. In many cancers, it is now recognized that ECM regulation plays a role in risk, subtype, progression, survival, recurrence, and drug resistance [115–123]. However, ECM presents a unique analytical challenge due to collagen triple helical structures that form suprastructural fibers, crosslinking networks, and extensive posttranslational modifications (PTMs) [124,125]. Typically, by conventional untargeted tryptic proteomics, ECM peptides make up a small percentage of spectral features in IMS data [124,126]. Decellularization of intact organs or tissue sections is an approach that has been used to enrich for ECM type peptides in IMS studies by proteomics after trypsin digestion [127]. This strategy identified localized elevation of COL3A1 peptides in the aorta compared to the rest of the heart. In addition, kidney glomeruli from human renal carcinoma were identified as having increases in fibronectin [127]. For FFPE-type tissue, as used for archival clinical storage, decellularization is not an option due to formalin-induced crosslinking between all proteins.

A new approach that uses FFPE tissues and does not require decellularization has been developed to target ECM proteins by IMS. Instead of trypsin or similar protease, this method uses collagenase type III, which reports peptides from 60 to 80 ECM proteins including multiple collagen types [16]. Breast tissue pathologies from 176 patient biopsies were investigated using collagenase type III digestion to target and enrich for ECM. A major finding was that ECM peptide signatures could distinguish between benign type breast pathologies such as fibroadenoma and inflammation as well as subtypes of intraductal lobular carcinoma and invasive ductal carcinoma [76]. An example of ECM imaging of an invasive ductal breast cancer tissue is shown in Fig. 17.4. Another study found that hydroxylated proline modifications from collagen triple helical regions could sensitively and specifically distinguish between normal lung tissue and low-grade lung adenocarcinoma [77], suggesting that the ECM may be a target for markers of early lung cancer. Other IMS strategies combine ECM targeting approaches with other IMS methods targeting molecules. For instance, N-glycan analysis was done prior to a collagenase type III to facilitate combined N-glycan and ECM mapping from the same tissue section [78]. This strategy also improved detection of collagen peptides from lung adenocarcinoma. Isobaric and near-isobaric peptide masses are very common in ECM imaging and most ECM studies couple MALDI sources with FT-ICR for increased mass resolution and accuracy. High-resolution accurate mass data is required to link peptide masses with high throughput sequencing data from parallel LC-MS/MS workflows. With the current recognition that ECM plays a critical role in prediction of disease risk and progression, new IMS studies targeting the ECM are expected to result in therapies and biomarkers for disease research.

The Oxygen Sensing and Effector Mechanisms of Hypoxic Pulmonary Vasoconstriction

In eukaryotic cells, mitochondria are acting as powerhouses using mainly O2 to convert nutrients into chemical energy in the form of adenosine triphosphate (ATP) that is used in processes including muscle contraction, ion transport, nerve impulse propagation, and chemical synthesis. A series of oxidation-reduction reactions involves the transfer of electrons from nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) across the electron transport chain (ETC). Because mitochondrial respiration continues even at very low O2 levels (∼7 mm Hg) to generate ATP, it is unlikely that variations in ATP concentrations trigger the hypoxic response.16 Ultimately, in the process of oxidative phosphorylation, O2 serves as the terminal electron acceptor of the ETC to form H2O, while, at multiple sites along the ETC, some leaking electrons react with O2, forming reactive oxygen species (ROSs), such as superoxide and hydrogen peroxide.

In pulmonary VSMCs, the most plausible mechanism todetect hypoxia is the mitochondrial PO2-sensitive ability tovary the production of diffusible ROSs and/or to alter the cytosolic redox state. Although controversies surround the issue on whether ROSs generated by the ETC decrease or increase in response to hypoxia, there is strong evidence that HPV is triggered by mitochondrial signals that launch a coordinated response involving membranar and sarcolemnal potassium and calcium channel receptors to increase cytoplasmic calcium concentrations.17 As proposed by Smith and Schumacker,17 mitochondrial ROSs (see Fig. 15.4) move to the cytosol, where they activate several pathways leading to intracellular calcium mobilization: (1) inhibition of Kv channels causing membrane depolarization, which promotes the opening of VGCC; (2) activation of phospholipase C (PLC) to generate diacyl glycerol (DAG) and inositol 1,4,5-triphosphate (IP3) that stimulate calcium release, respectively, from membranal receptor-operating channels (ROCs) and sarcolemnal IP3 receptors; and (3) activation of ryanodine receptors (RyR) by oxidation of cysteine residues. In addition, by increasing the ratio of adenosine monophosphate (AMP) to ATP, low O2 tension stimulates AMP-kinase (AMPK), resulting in activation of the RyRs and further Ca2+ release from the sarcoplasmic reticulum (SR). Together, the elevation of intracellular Ca2+ concentrations induces calcium binding to calmodulin, subsequent activation of myosin light chain (MLC) kinase, myosin phosphorylation, and ultimately contractions of VSMCs.

After activation of Rho kinase and MLC phosphatase, myofibrillar proteins become more sensitized to the effects of calcium sensitization, resulting in sustained contractions of pulmonary VSMCs. Besides acting directly on calcium channels, ROSs influence the cytoplasmic redox state, namely, the ratio of oxidized to reduced proteins, glutathione (GSSG/GSH), and nicotinamide adenine dinucleotide (NAD/NADH). According to the redox hypothesis, hypoxic conditions are associated with lesser release of ROSs from the mitochondria that promotes a shift from an oxidized to reduced cellular redox state in the cytoplasm, which in turn triggers Ca2+ mobilization through inhibition of Kv channels and opening of VGCCs.

When exposed to hypoxia, both pulmonary and systemic VSMCs share the same effector mechanisms of calcium-mediated changes in vascular tone, whereas pulmonary VSMCs present the unique property to detect low levels of O2 tension and transmit signals to mobilize Ca2+ close to myofibrillar proteins. Mitochondria are located closer to the plasmalemmal membrane in pulmonary VSMCs compared with systemic VSMCs.18 Therefore a greater structural and functional coupling between mitochondria and Kv channels in pulmonary VSMCs could explain the specific O2 sensitivity and HPV response.

Phosphate Transport and Osteogenic Cell Function

Like all eukaryotic cells, osteogenic cells (i.e., osteoblasts and chondrocytes) are endowed with Na-dependent Pi transport systems driven by an inwardly directed Na gradient. There are several differences between Na-dependent Pi transport in osteoblasts and that in renal epithelial cells. Affinity constants for Pi are significantly higher in osteoblasts (300–500 vs 50–100 µ M for renal epithelial cells) and Na/Pi cotransport in osteoblasts is stimulated by an acidic extracellular pH [35], whereas renal epithelial Pi transport is stimulated by more alkaline pH. PTH, a potent osteotropic factor, stimulates Na/Pi cotransport in osteoblasts [136] but inhibits transport in renal proximal cells. Additional studies are necessary to understand the cellular and molecular mechanisms underlying these differences.

Other factors that stimulate Na/Pi cotransport in osteoblasts, such as insulin-like growth factor-1 (IGF-1), platelet-derived growth factor, and fibroblast growth factor (FGF), also enhance the proliferation and/or the differentiation of bone-forming cells [103,119,143,172]. The cellular mechanisms by which these growth factors increase Pi transport in osteogenic cells are not completely understood but may involve a tyrosine phosphorylation process [32]. In addition, fluoride, another potent activator of bone formation, also stimulates Pi transport in osteoblastic cells via the activation of a tyrosine phosphorylation process [34].

Oxidation

DNA of eukaryotic cells is continuously subjected to reactive oxygen species (ROS) exposure which can be derived from the external environment, but predominantly from endogenous byproducts of the oxidative phosphorylation events which occur during mitochondrial respiratory chain of all aerobic organisms.8 Another major source of ROS production is represented by phagocytic NADPH oxidases during inflammatory responses and by non-phagocytic NADPH oxidases, as determined in different cell systems.9 In its ground state, molecular oxygen (O2) is relatively unreactive. However, during normal metabolic activity, and as a consequence of various environmental perturbation (e.g. radiation, biotic, and abiotic stresses, xenobiotics and diseases) O2 is capable of giving rise to frightfully reactive excited states such as free radicals and derivatives.10 All ROS are extremely reactive and can cause molecular damage, leading to cell death.11 Purines undergo oxidation of the ring atoms, leading to various chemical modifications. The highly mutagenic guanine derivate 8-hydroxyguanine (8-oxoG) is formed in large quantities as a consequence of the high oxidation potential of this base (Figure 3.1).12 The miscoding effect of 8-oxoG lesion is due to DNA polymerase activity which inserts adenine opposite to 8-oxoG, resulting in G:C to A:T transition mutations, therefore generating a DNA base mutation.

The most frequent pyrimidine oxidation is represented by the formation of 5-hydroxycytosine (5-OHC) which leads to the insertion of a thymine creating a potential premutagenic lesion.13 Other important oxidation lesions are the formamidopyrimidine such as faPyA and faPyG and the oxidized thymine glycol (TG) (see Figure 3.1).14 All these mutations are recognized by different DNA glycosylases, which remove the damaged nitrogenous base by cleaving the N-glycosylic bond and generating an abasic (AP) site while leaving the sugar-phosphate backbone intact. This reaction represents the initiation step of the BER pathway and forms the substrate for the AP endonuclease enzyme which generates a nick in the phosphodiester backbone of the AP site.

5.2.1.2 Targeted gene modification

In many eukaryotic cells, NHEJ-mediated gene disruption via the CRISPR/Cas9 system is a powerful tool used for functional gene analysis. However, when used in S. cerevisiae, the efficacy of this method was low compared with other eukaryotes (0.5–1.1%) (Gao et al., 2016; Morse et al., 2018) (Table 5.1). Meanwhile, highly efficient HR-mediated gene modification, targeted gene disruption, deletion, single allele swapping, and base substitution could be performed using the CRISPR/Cas9 system and donor DNA fragments (25–100%) (Table 5.1). However, when donor DNA fragments do not exist in the cells, repetitive DSB introductions via the CRISPR/Cas9 system lead to negative effects. Therefore, the CRISPR/Cas9 system enables marker-free genome editing in S. cerevisiae, and manipulation of multiple alleles with high efficiency in diploid (30–100%) and polyploid (15–60%) industrial yeasts (Table 5.1).

Recently, other Cas9 variants, orthologs, or different types of CRISPR/Cas system have been characterized and used for more flexible genome editing (Mitsunobu et al., 2017). PAM requirements and nuclease features between the CRISPR/Cas9 systems of various bacterial species such as the Cas9 from Neisseria meningitides (NmCas9, NNGRRT PAM) (Hou et al., 2013) and Streptococcus thermophiles (St1Cas9, TTTN PAM) (Esvelt et al., 2013; Kleinstiver et al., 2015) vary. Except for class 2/type II CRISPR/Cas system, CRISPR/Cpf1 (Cas12a) (Zetsche et al., 2015), which belongs to class 2/type V, has also been used as an alternative genome editing tool in several organisms. CRISPR/Cpf1 system recognizes thymidine-rich PAM, such as the Cpf1 form Lachnospiraceae bacterium ND2006 (LbCpf1, TTTN PAM), Acidaminococcus sp. BV3L6 (AsCpf1, TTTN PAM) and Francisella novicida (FnCpf1, TTTN PAM) located at the 5′ end of target region, and can be recruited by a single crRNA with self-processing activity without a tracrRNA (Zetsche et al., 2015). In S. cerevisiae, NmCas9 and AsCpf1 were nonfunctional while St1Cas9, LbCpf1, and FnCpf1 were functional, although these Cas9 variants and orthologs are functional in mammalian cells (Lian et al., 2017; Verwaal et al., 2018; Swiat et al., 2017). These Cas9 variants and orthologs can expand yeast genome editing, but optimization of these systems for yeasts needs codon-usage and addition of the NLS sequence at the N- and C-terminus of Cas proteins.

Eukaryotic Expression Vectors

Because transfection of eukaryotic cells is a relatively inefficient process it is necessary to use high concentrations of DNA. Production of large quantities of DNA is most easily accomplished by cloning the coding region of the protein of interest into a plasmid, transforming suitable bacteria, and isolating the plasmid DNA from the bacteria. Thus, it is important, if possible, to select a vector that grows to high copy number in bacteria. A number of other factors must also be considered when selecting a eukaryotic expression vector. The requirements for the vector will vary depending on the experimental design. If transient expression of a protein is desired the most important consideration is that the coding region of the protein be inserted in the proper transcriptional orientation with respect to the promoter. A critical consideration regardless of whether one intends to stably or transiently transfect cells is the promoter. The promoter must be a eukaryotic promoter that is efficient in the cells of choice. We have found that the Rous Sarcoma LTR is not very efficient in Hep G2 cells. Conversely, we have found that the cytomegalovirus immediate early promoter enhancer is very efficient in Hep G2 cells. Additionally, the vectors that are used for the establishment of stably transfected cells must contain a marker, under the control of an eukaryotic promoter, that can be selected for in eukaryotic cells. Typically, the selectable marker used is an antibiotic resistance factor. Among the antibiotic resistant factors commonly used are genes that confer resistance to antibiotic such as zeocin, hygromycin B or neomycin (G418).

For constitutive expression of proteins in Hep G2 cells we have used pcDNA3.1 (InvitroGen, Carlsbad, CA, USA). A variety of eukaryotic expression vectors are commercially available and should be chosen with care, depending on the experimental design.