1.4: Tour of the Eukaryotic Cell

Here we take a closer look at the division of labors among the organelles and structures within eukaryotic cells. We’ll look at cells and their compartments in a microscope and see how the organelles and other structures were isolated from cells and identified not only by microscopy, but by biochemical and molecular analysis of their isolates.

1.4.1. The Nucleus

The nucleus is the largest organelle in the cell, separating the genetic blueprint (DNA) from the cell cytoplasm. Although the eukaryotic nucleus breaks down during mitosis and meiosis as chromosomes form and cells divide, it spends most of its time in its familiar form during interphase, the time between cell divisions. The structural organization of an interphase nucleus is shown in Figure 1.11 below.

Figure 1.11: LEFT: Transmission electron micrograph of an insect cell nucleus showing the nuclear envelope(ne) and nucleolus(n);

RIGHT: Drawing of a nucleus with chromatin (purple) and nuclear pores

The cross-section of the interphase nucleus in the electron micrograph shows a prominent nucleolus (labeled n). The nucleus is enclosed in a nuclear envelope and surrounded by a darkly granular RER (rough endoplasmic reticulum). You can make out ribosomes (small granules) bound to the RER and to the outer nuclear membrane.The space enclosed by the RER (the lumen) is in fact continuous with the space separating the inner and outer membranes of the nuclear envelope, as illustrated in the drawing (above right). Nuclear pores in the nuclear envelope (look at the drawing) let large molecules and even particles move in and out of the nucleus across both membranes. The eukaryotic nucleus is where genes and RNA transcription are regulated and thus one place where cellular protein levels are controlled. RNAs, once transcribed from genes and processed, are exported to the cytoplasm through the nuclear pores. Even completely assembled ribosomal subunits are exported from the nucleus. Other RNAs remain in the nucleus, often participating in the regulation of gene activity. We learn some details of nuclear pore traffic, DNA replication, and the dynamics of cell division in later chapters.

104-2 The nucleus

Beyond its nucleolus and nuclear envelope, the nucleus is more organized than it appears in conventional transmission electron micrographs.The nucleolus is just the largest of several inclusions that seem to segregate nuclear functions. Over100 years ago Santiago Ramón y Cajal reported other structures in the nuclei of neurons, including what came to be known as Cajal bodies (CBs). His elegant hand-drawn illustrations of nuclear bodies (made before the advent of photomicrography) can be seen at Cajal's Nuclear Bodies and Cajal'sBeautiful Brain Cells. Cajal and Camillo Golgi shared the Nobel Prize in Physiology or Medicine 1906 for their studies of nerve cell structure. In the electron microscope, Cajal bodies (CBs) look like coils of tangled thread, and were thus called coiled bodies (conveniently, also CBs). Other nuclear bodies since identified include Gems, PML bodies, nuclear speckles (or splicing speckles), histone locus bodies (HLBs), and more! The results of immunofluorescence localization studies show that different nuclear bodies are associated with specific proteins (Figure1.12, below).

Figure 1.12: Simulated immunolocalization of different proteins (fibrillarin, coilin and ASF/SF2) to nuclear bodies (nucleolus, Cajal Bodies and nuclear speckles, respectively), using fluorescent antibodies

Nucleoli contain fibrillarin proteins, stained red by treating cells with red-fluorescence-tagged antibodies to fibrillarin. Pink-fluorescence-tagged anticoilin antibodies light up the coilin proteins of CBs. Green-fluorescing ASF/SF2 antibodies localize to nuclear speckles. As part of or included in a nuclear matrix, nuclear bodies organize and regulate different aspects of nuclear activity and molecular function. The different nuclear bodies perform specific functions and interact with each other and with proteins DNA and RNA to do so. We will revisit nuclear bodies in their working context later.

1.4.1.a Every Cell (i.e., Every Nucleus) of an Organism Contains the Same Genes

We read earlier that bacteria are busy doubling and partitioning their naked DNA chromosomes at the same time as they grow and divide by binary fission. In eukaryotic cells, a cell cycle divides life into discrete consecutive events. During most of the cell cycle, cells are in interphase and DNA is wrapped up in proteins in chromatin inside a nucleus. It is not merely the DNA, but chromatin that must be duplicated when cells reproduce. Duplication of DNA also involves disturbing and rearranging the chromatin proteins resting on the DNA. This occurs before cell division. As the time of cell division nears, chromatin associates with even more proteins, condensing to form chromosomes, while the nuclear envelope dissolves, marking the start of mitosis (meiosis in germline cells) and cytokinesis. You may recall that each somatic cell of a eukaryotic organism has paired homologous chromosomes and thus two copies of every gene the organism owns. But sperm and eggs emerge from meiosis with one of each pair of chromosomes and only one copy of each gene. Whether by mitosis or meiosis, duplicated chromosomes(chromatids) lined up at metaphase attach to spindle fibers (as seen in Figure 1.13) to be separated and drawn into new daughter cells formed during cytokinesis.

Figure 1.13: Drawing (left) and fluorescence micrograph (right) of a cell in metaphase of mitosis: aligned chromosomes (chromatids) at the center of the cell (blue in the micrograph) are just about to be pulled apart by microtubules of the spindle apparatus (green) extending from the poles to the center of the cell.

As chromosomes separate and daughter cells form, nuclei reappear and chromosomes de-condense. These events mark the major visible difference between cell division in bacteria and eukaryotes. Cytokinesis begins near the end of mitosis. Sexual reproduction, a key characteristic of eukaryotes, involves meiosis rather than mitosis. The mechanism of meiosis, the division of germ cells leading to production of sperm and eggs, is like mitosis except that the ultimate daughter cells have just one each of the parental chromosomes, eventually to become the gametes (eggs or sperm). Google meiosis and/or mitosis to remind yourself about the differences between the two processes, meiosis and mitosis. A key take-home message here is that every cell in a multicellular organism, whether egg, sperm or somatic, contains the same genome (genes) in its nucleus. This was already understood from the time that mitosis and meiosis were first described in the late nineteenth century.

That every cell of an organism really does contain copies of all of its genes was finally demonstrated by John Gurdon and Shinya Yamanakain 1962. They transplanted nuclei from the intestinal cells the frog Xenopus laevis into enucleated eggs (eggs from which their own nuclei had been removed). These ‘eggs’ grew and developed into normal tadpoles, proving that no genes are lost during development, but are just expressed differentially. For these cloning experiments, Gurdon and Yamanaka shared the 2012 Nobel Prizefor Physiology or Medicine. We’ll revisit animal cloning later. For now, it’s enough to know that Molly the cloned frog was followed by Dolly, the first cloned sheep(1966) and then other animals, all cloned from enucleated eggs transplanted with differentiated cell nuclei. See Cuarteterra to read about the cloning a champion polo mare whose clones are also champions

CHALLENGE

One group of bacteria (Planctomycetes) does in fact surround their nucleoid DNA with a membrane! How do you think these cells divide their DNA equally between daughter cells during cell division?

1.4.2. Ribosomes

On the tiny end of the size spectrum, ribosomes are protein-making machines found in all cells. They consist of large and small subunits, each made up of proteins and ribosomal RNAs (rRNAs). Ribosomes bind to messenger RNAs (mRNAs), moving along the mRNA to translate 3-base code words (codons) into polypeptides. Multiple ribosomes can move along the same mRNA, forming polyribosomes (or polysomes) that simultaneously translate the same polypeptide encoded by the mRNA as shown in Figure 1.14.

Figure 1.14: Polysomes form when ribosomes (blue) assembled at the left on an mRNA molecule move.As they move from left to right more ribosomes can assemble at the left, each translating the message into a polypeptide (green) that grows and emerges from the ribosomes.To accommodate ribosome size and the allosteric (shape) changes during polypeptide synthesis, there are at least 35 nucleotides of separation between ribosomes on polysomes.

In the illustration, ribosomes assemble on the left (5’) end of the messenger RNA to form the polysome. When they reach the other (3’) end of the mRNA, the ribosomes disassemble from the RNA and release the finished polypeptide. The granular appearance of cytoplasm in electron micrographs is largely due to the ubiquitous distribution of ribosomal subunits and polysomes in cells. In electron micrographs of leaf cells from a dry, desiccation-tolerant dessert plant, Selaginella lepidophylla (Figure1.15), you can make out randomly distributed ribosomes and ribosomal subunits (arrows, below left). In cells from a fully hydrated plant, you can see polysomes as more organized strings of ribosomes (arrows, below right).

Figure 1.15: Transmission electron micrographs of Selaginella lepidophylla plant cells. Small,free ribosomal subunits in desiccated cells (left) seem to have formed larger structures (ribosomes) aligned on polysomes in the hydrated plant cells (right). Zoom in to see these details.

Isolated ribosomes and subunits can be separated by sucrose-density-gradient centrifugation based on differences in mass. Figure1.16 compares ribosomal subunit ‘size’, protein, and ribosomal RNA (rRNA) composition in eukaryotes and prokaryotes.

Figure 1.16: Comparison of prokaryotic (upper) and eukaryotic (lower) ribosomal subunit, rRNA and protein composition. The subunits and their component rRNAs were separated by mass (i.e., size) by sucrose density gradient ultracentrifugation. Proteins were isolated from separated subunits for analysis.

S (Svedberg) units are calculated from the position of particles and molecules in the gradient after separation. Theodor Svedberg earned the 1926 Nobel Prize in Chemistry for among other things, applying analytical ultracentrifugation to the separation and determination of particulate and molecular masses.

101 Ribosomes & Polysomes

1.4.3. Internal membranes and the Endomembrane System

Microscopists of the nineteenth century saw many subcellular structures using the art of histology, staining cells to increase the visual contrast between cell parts. One of these microscopists was the early neurobiologist, Camillo Golgi. He developed a silver (black) stain that first detected a network of vesicles which we now call Golgi bodies (or Golgi vesicles) in nerve cells. For his studies of the membranes now named after him, Camillo Golgi shared the 1906 Nobel prize for Medicine or Physiology with Santiago Ramón y Cajal.

Golgi vesicles along with other vesicles and vacuoles in cells, including, comprise the endomembrane system. Proteins made by ribosomes of the rough endoplasmic reticulum (RER) either enter the interior space (lumen) or become part of the RER membrane itself. The syntheses of RER, smooth endoplasmic reticulum (SER), Golgi bodies, microbodies, lysosomes, and other vesicular membranes (and their protein content) all start in the RER. transport vesicles that bud off from RER fuse with Golgi Vesicles at their cis face (Figure1.17).

Figure 1.17: Transmission electron micrograph of an insect cell Golgi body showing cis and trans faces (labeled in white). Vesicles from the RER at the cis face (below, right) will fuse with the Golgi vesicles.Vesicles emerging at the trans face (above, left) are progenitors of a variety of different organelles.

Some proteins made in the endomembrane system are secreted by exocytosis. Others end up in organelles such as lysosomes that contain hydrolytic enzymes. These enzymes are activated when the lysosomes fuse with other organelles destined for degradation. For example, food vacuoles form when a plasma membrane invaginates, engulfing food particles. They then fuse with lysosomes to digest the engulfed nutrients. Still other proteins synthesized by ribosomes on the RER are incorporated into the RER membranes, destined to become part of lysosomes, peroxisomes, and even the plasma membrane itself. In moving through the endomembrane system, packaged proteins undergo stepwise modifications (maturation) before becoming biologically active (Figure1.18, below).

Figure 1.18: Illustration of ‘packaged’ protein traffic through a cell from the RER (lower left) through Golgi vesicles (middle) to organelles (e.g., lysosomes) and the plasma membrane (upper right) for exocytosis(i.e., secretion). RER and Golgi vesicles are major sites for the modification (i.e., maturation) of packaged protein.

100-2 The RER-Rough Endoplasmic Reticulum

102 Golgi Vesicles & the Endomembrane System

Autophagosomes are small vesicles that surround and eventually encapsulate tired organelles (for example, worn out mitochondria), eventually merging with lysosomes whose enzymes degrade their contents. In 2016, Yoshinori Ohsumi earned the Nobel Prize in Physiology and Medicine for nearly 30 years of research unraveling the cell and molecular biology of autophagy. Microbodies are a class of vesicles smaller than lysosomes but formed by a similar process. Among them are peroxisomes that break down toxic peroxides formed asa by-product of cellular biochemistry. Some vesicles emerging from the RER lose their ribosomes to become part of the SER, which has several different functions (e.g., alcohol detoxification in liver cells).

103-2 Smooth Endoplasmic Reticulum

Other organelles include the contractile vacuoles of freshwater protozoa that expel excess water that enters cells by osmosis. Some protozoa have extrusomes, vacuoles that release chemicals or structures that deter predators or enable prey capture. A large aqueous central vacuole dominates the volume of many higher plant cells. When filled with water, they will push all other structures against the plasma membrane. In a properly watered plant, this water-filled vacuole exerts osmotic pressure that among other things keeps plant leaves from wilting and keeps stems upright

1.4.4. Mitochondria and Plastids

Nearly all eukaryotic cells contain mitochondria, shown in Figure 1.19.

Figure 1.19: Transmission electron micrograph of mitochondria embedded in RER (left) and a labeled drawing of a single mitochondrion (right).

The matrix of the mitochondrion is enclosed by a cristal membrane surrounded by an outer membrane. Each contains and replicates its own DNA, which contains genes encoding some of the mitochondrial proteins. The surface area of the inner mitochondrial membrane is increased by being folded into cristae, which are sites of cellular respiration (aerobic nutrient oxidation). Later, we’ll consider the role of mitochondria in respiration in more detail.

Earlier, we speculated that some eukaryotic organelles could have originated within bacteria. But mitochondria probably evolved from anaerobic bacterium that was engulfed by another cell that escaped destruction to become an endosymbiont in the host cell. Lynn Margulis first proposed this in her Endosymbiotic Theory, in which a primitive eukaryotic cell acquired a bacterial endosymbiont (Margulis, L.[Sagan, L], 1967, On the origin of mitosingcells. Journal of Theoretical Biology 14: 225–274). She proposed that chloroplasts also started as endosymbionts. Both mitochondria and the plastids of plants contain their own DNA, transcribe it into RNA and use their own translational machinery (i.e., ribosomes) to synthesize proteins, further supporting their bacterial and cyanobacterial origins. Living at first in symbiosis with the rest of the cell, these endosymbionts would eventually evolve into the organelles that we are familiar with.

Several protozoa lacking mitochondria and other organelles were discovered and suggested to be “first ingestors” of an ancestral endosymbiont, but since these cells contain other organelles (e.g., hydrogenosomes, mitosomes) it is thought more likely that these species once had, but then lost mitochondria.

Therefore, the descendants of ancient eukaryotic cells missing mitochondria probably no longer exist, if they ever existed at all! More evidence for the Endosymbiotic Theory is discussed elsewhere.

CHALLENGE

Nick Lane favors an endosymbiotic event where one prokaryote engulfed another prokaryote (Mitochondria evolve from Bacterium-in-Bacterium Endosymbiosis). What is the dramatic, unorthodox consequence to evolutionary thought if Lane is right about this?

Chloroplasts, photosynthetic protozoa, and cyanobacteria contain chlorophyll and use similar photosynthetic mechanisms to make glucose. Transmission electron micrographs of chloroplasts are shown in the Figure 1.20. The one on the right shows a few starch granules.

Figure 1.20: Transmission electron micrographs of a typical photosynthetic chloroplast (LEFT), and one that has photosynthesized long enough to accumulated starch granules (RIGHT). S, starch granule; T, thylakoids

A leucoplast is also a plastid, a chloroplast that has become filled with starch granules. In the electron micrograph of a leucoplast in Figure1.21, you can see that, because of the accumulation of starch, the grana have become dispersed and indistinct.

Figure 1.21: Electron micrograph of a leucoplast, a chloroplast that has become filled with starch granules (S).

105-2 Endosymbiosis-Mitochondria & Chloroplasts

1.4.5. Cytoskeletal structures

We have come to understand that the cytoplasm of a eukaryotic cell is highly structured, permeated by rods and tubules. The three main structural components of this cytoskeleton are microfilaments, intermediate filaments,and microtubules.The structure and polarity of these structures are shown in Figure 1.22.

Figure 1.22: Assembly, structure and polarity of microtubules (upper), microfilaments middle), and intermediate filaments (lower) with electron micrographs of isolated microtubules upper right) and immunofluorescence micrographs of using fluorescent antibodies to microfilament and intermediate filament proteins (middle and bottom right, respectively)

Microtubules are composed of \(\alpha\)− and \(\beta\)−tubulin protein monomers. Monomeric actin proteins make up microfilaments. Intracellular intermediate filament proteins are related to the extracellular keratin of hair, fingernails, claws, and bird feathers. These cytoskeletal rods and tubules not only determine cell shape, but also play a role in cell motility. This includes the movement of cells from place to place and the movement of structures within cells.

We have already noted that a prokaryotic cytoskeleton is composed in part of proteins homologous to the actins and tubulins. As in a eukaryotic cytoskeleton, these bacterial proteins may play a role in maintaining or changing cell shape. On the other hand, flagellin(a protein not found in eukaryotic cells) powers the movement of bacterial flagella.

A bacterial flagellum is a rigid hook-like structure attached to a molecular motor in the cell membrane that spins to propel the bacterium through a liquid medium. In contrast, eukaryotic microtubules slide past one another causing a more flexible flagellum to undulate in wave-like motions and a cilium to beat rather than undulate. Cilia are involved not only in motility, but also in feeding and sensation. Microtubules in eukaryotic flagella and cilia arise from a basal body (similar to kinetosomes or centrioles) such as the one in Figure 1.23.

Figure 1.23: Transmission electron micrograph of a longitudinal section of a flagellum (#1) emerging from a basal body (2). Number 3 is another basal body, this time in cross section.

Aligned in a flagellum or cilium, microtubules form an axoneme surrounded by plasma membrane. In electron micrographs of cross sections, a ciliary or flagellar axoneme is typically organized as a ring of nine paired microtubules (called doublets) around two singlet microtubules. Figure 1.24 shows the \(9+2\) microtubule arrangement of an isolated axoneme.

Figure 1.24: Drawing of an axoneme isolated from a eukaryotic cilium or flagellum, by removing their plasma membrane, showing their characteristic \(9+2\) arrangement of microtubules in cross section (at the left) and longitudinal section projecting to the right.

Centrioles are themselves comprised of a ring of microtubules. In animal cells they participate in spindle fiber formation during mitosis and meiosis and are the point from which microtubules radiate thorough the cell to help form and maintain its shape. These structures do not involve axonemes. The spindle apparatus in plant cells typically lack centrioles but form from an amorphous MTOC, or MicroTubule Organizing Center. The MTOC serves the same purpose in mitosis and meiosis as centrioles serve in animal cells.

106-2 Filaments & Tubules of the Cytoskeleton

Elsewhere, we describe how microfilaments and microtubules interact with motor proteins (e.g., dynein, kinesin, and myosin) to generate force that results in the sliding of filaments and tubules to allow cellular movement. You’ll see that motor proteins also transport molecular cargo from one place to another in a cell.