BOTANY VOLUME 1 - CELL BIOLOGY. ANATOMY. MORPHOLOGY - 2007

2. CELL STRUCTURE AND ULTRASTRUCTURE

The Structure AND Functions of The Cell constitute the Subject Matter of cell biology. This field encompasses The Study of ultrastructure, biochemistry, and molecular biology, as well as many aspects of cellular physiology. Until 1950, prior to the advent of modern Research Methods at THE CELLULAR LEVEL, the Study of the cell was known as Cytology (from Greek kytos — pouch, cell). To a large extent, its capabilities were limited by light Microscopy.

The Significance of studying the cell lies in the fact that all Living organisms are composed of Cells. Many organisms are unicellular: a single cell represents an entire individual. This is observed in the majority of prokaryotes and, by definition, in all eukaryotic protists—including, for example, flagellates from various algal divisions, as well as diatoms. Admittedly, in terms of species number, eukaryotes are dominated by Multicellular Organisms. Because cells are overwhelmingly microscopic in size, their number in large multicellular organisms is often unimaginably large. A single tree may consist of more than 10,000 billion (10¹³) cells. A single medium-sized leaf of an angiosperm is constructed from approximately 20 million (2∙10⁷) cells. SHOOT and ROOT apical Meristems contain from 1,000¹ to 500,000 cells capable of division.

¹ Sometimes meristems consist of a small number of cells; for instance, in the model plant Arabidopsis thaliana, the root meristem contains an order of magnitude fewer cells. — Ed. note.

Multicellular organisms are evolutionarily younger than unicellular ones. Over the course of the evolution of multicellular organisms, essential life processes have remained unchanged at the level of the individual cell. This primarily concerns nutrient storage, reproduction, and the expression and recombination of Genetic information. Almost every cell in an Organism contains a complete, and most frequently even double (diploid) set of genes and, accordingly, Chromosomes. A cell can

duplicate this set of genes through METABOLISM/36.html">DNA Replication and pass it on in precisely equal parts to daughter cells (mitosis) (see 2.2.3.5). Cells of a multicellular organism generally possess the same set of genes—they belong to a single Gene clone. The fact that they nevertheless differentiate in a regular manner during individual development (ontogeny)—i.e., acquire distinct morphologies and perform different functions—appears, at first glance, paradoxical under such circumstances. However, The problem of differentiation and, correspondingly, determination is now fundamentally resolved, as it has been established that a specific state of differentiation always corresponds to the Activation of a defined specific portion of The Genome and the suppression of the remaining genes. Gene activation and suppression are governed by integration signals which (since they do not originate from the environment and do not mediate individual adaptations) ultimately, within a multicellular system, also emanate from cells and are perceived by other, specialized cells.

Processes associated with sexual reproduction cannot occur without the participation of individual cells. For this purpose, specialized Germ Cells—Gametes—are typically formed. Biologically significant processes associated with sexual reproduction include Meiosis with the recombination of hereditary information, as well as syngamy—the fusion of cells and nuclei from gametes of the same species that are nevertheless genetically non-identical (see 2.2.3.7 — 2.2.3.9).

Cells can arise only from preexisting cells through division or fusion: "omnis cellula e ex cellula" (R. Virchow, 1855)¹. The characteristics of living systems listed in the textbook Introduction collectively manifest themselves only at the cellular level, and not below. The cell has thus proven to be the smallest viable unit—the elementary organism. This also applies to multicellular organisms, as confirmed by the aforementioned phenomena of sexual processes, such as The ability to maintain individual cells in culture (Fig. 2.1).

¹ "Every cell [derives] from a cell" (Lat.) — this principle, formulated in 1855 by R. Virchow, remains a foundational tenet of Cell Theory to this day. — Transl. note.

Class="center">Fig. 2.1. One-week-old suspension culture of soybean cells (Glycine max, 90x) (cell culture — H. Grisebach, K. Hahlbrock, LM micrograph — N. Falk)

Some cells have already divided once or several times; eventually, a whole plant can grow back from each cell, which is utilized for cloning, i.e., for the artificial production of genetically uniform plant material

2.1. The Study of the Cell

The history of The Development of cell study methods is a prime example of how scientific progress depends on technological capabilities. Cells are for the most part microscopically small.

The first descriptions of cells appeared in the 17th century, following the invention of the first microscope. However, the fundamental similarity between plant, animal, and protist cells was established only after the microscope was significantly improved by the beginning of the 19th century. Following the Discovery of the Cell Nucleus in plant Tissues, its presence in animal and human cells also became evident. In 1839, T. Schwann published his epoch-making work, "Microscopic Researches Concerning the Accordance in the Structure and Growth of Animals and Plants" (T. Schwann, Mikroskopische Untersuchungen über die Übereinstimmung in der Struktur und dem Wachstum der Thiere und Pflanzen). This laid the foundations of general biology. Gradually, as microscopic observation capabilities continually expanded and As a result of early investigations in cellular physiology (such as osmosis), further discoveries followed.

In the second half of the 19th century, three core principles of cytology became increasingly clear.

✵ All living organisms are constructed of cells.

✵ Many organisms are unicellular.

✵ The individual development of multicellular organisms begins—at least in sexual reproduction—with a unicellular stage.

Around the 1880s, as a result of a major new refinement in microscopic optics carried out by E. Abbe, the maximum theoretically possible resolution of 0.2 µm was achieved for the first time. Simultaneously, notable progress was made in the preparation techniques for microscopy.

By 1900, virtually all cellular Organelles visible under light microscopy had been described (Fig. 2.2).

Fig. 2.2. Plant cell under the Light Microscope (A—after D. von Denffer; B, C—Interference contrast; D—phase contrast): A—diagram of a cell from the mesophyll parenchyma of a higher plant leaf; B—Chloroplasts in the leaf cells of a bryophyte moss (Mnium undulatum, 300x); C—epidermal Cells of the onion (Allium cepa) in interference contrast (130x): the large cells are almost entirely filled with a central vacuole; the peripheral layer of Cytoplasm, which is thickened at the corners, contains The Nucleus with a nucleolus; D—nuclear region of an Allium cell as in C, phase contrast (3100x); Chromatin and nucleolus are visible in the nucleus, while elongated Mitochondria and rounded oleosomes are seen in the cytoplasm alongside leucoplasts (two of which contain clear starch inclusions)

Following the rediscovery of G. Mendel's Laws of inheritance at the turn of the 20th century, primary attention shifted over the next 40 years to the nucleus and chromosomes (karyology, cytogenetics).

The explosive development of cell research after 1945—in the fields of ultrastructure, biochemistry, and molecular biology—was once again driven by technological progress: the invention of the Electron microscope, the development of Cell Fractionation using ultracentrifugation (Box 2.1), and X-ray structure analysis of biological macromolecules. Recently, fundamentally new observation and preparation techniques have vastly expanded the possibilities for studying living cells. This is of particular importance in the transition from the era of Genomics to the era of Proteomics.

2.1.1. Light Microscopy

The objective of a light microscope (LM) (Fig. 2.3) produces (much like a slide projector lens) an enlarged image of the specimen (preparation), which can be photographed. This intermediate image is viewed through the eyepiece acting as a magnifying Glass. The smallest, still distinguishable details of a specimen must be at a distance of at least 0.2 µm (200 nm) from each other. Macromolecular structures of the cell remain unresolved. Nevertheless, the LM retained its importance even after the advent of the electron microscope, which has a significantly higher resolution. It allows objects to be observed in a living state and requires considerably less effort in specimen preparation.

Since cellular structures are mostly colorless and differ only slightly from one another in light refraction, they often remain invisible even when their dimensions exceed the resolution limit. Accordingly, classical light microscopy primarily investigates fixed (killed while preserving structure) and artificially stained preparations. Optically anisotropic cellular structures, such as cell walls, starch grains, and nuclear division spindles, can also be observed in living cells using a polarizing microscope, which also allows for the analysis of their macromolecular architecture. Today, the contrast problem has been solved through optical manipulations that do not affect the specimen itself. Using phase contrast or differential interference contrast (DIC), phase differences of light waves passing through the specimen are transformed into contrast differences or a relief-like image (see Figs. 2.2C, D; 2.81; 3.9). Especially delicate cellular structures can be made visible through digital photography and Image Processing. This material is then further processed using computer technology—video microscopy. Three-dimensional reconstruction of cellular structures is made possible by confocal laser scanning microscopy (CLSM). This involves microscopic tomography: the intact, unaltered specimen is viewed as a series of ultra-thin optical "sections," from which a computer then generates a virtual spatial image. This volumetric reconstruction of a living object can be viewed on screen from any angle. In individual optical section images (which are constructed on a raster basis, i.e., line by line, much like in a movie), microscopic details are usually resolved much better than in the specimen itself, as interfering overlays are removed through data processing.

Fig. 2.3. Modern research light microscope (Axioplan, Carl Zeiss)

A—side view from the left, B—optical path. 1, 2—illumination for viewing material in transmitted light and against a dark Background, 3—fine focusing knob for the stage, 4, 5—condenser for illuminating the phase-contrast and DIC light field, 6—revolving nosepiece with objectives, above it a slot for light and polarizing filters and other optical attachments, 7—binocular tube, 8—automatic camera for photomicrography, 9—eyepiece

Cytochemical methods serve to identify and localize specific molecules within the cell. Among these, highly sensitive fluorescence methods play a crucial role. In a fluorescence microscope, the specimen is illuminated with short-wavelength light, causing the corresponding substances within the specimen to be excited and emit longer-wavelength quanta (fluorescence). To produce an image, the specimen is viewed through an optical filter that blocks the excitation radiation, so that only the fluorescent PARTS OF THE object remain illuminated. Since very few cell components naturally fluoresce strongly, a range of techniques has been developed for the specific fluorescent staining of particular molecules. Immunofluorescence is of particular importance in this regard. It is based on the extreme Specificity of mammalian immune system Antibodies, which allows for the precise Intracellular Localization of various Proteins, Polysaccharides, or Nucleic Acids acting as Antigens (e.g., Fig. 2.10)¹. Over the past decade, The Use of green fluorescent protein (GFP) as a fluorescent marker has expanded rapidly. It reveals gene activity in living cells and, for certain proteins, also makes it possible to study their appearance, localization, and behavior (cf. Fig. 2.83C).

¹ The Staining Procedure consists of several steps: 1. Application of antibodies against a specific antigen to the specimen, resulting in specific binding. 2. Washing away antibodies that have not bound to the specimen. 3. Application of secondary antibodies with covalently attached fluorescent molecules. The secondary antibodies react with the primary antibodies immobilized on the cellular structures. 4. Washing away unbound secondary antibodies. Only after all these Procedures is the specimen examined under the microscope. Such a complex methodology requires numerous controls, without which the distribution of the studied antigens in the cell cannot be reliably assessed. — Ed. note.

Рис. 2.4. Микрорадиоавтограмма (В—Е — препараты и съемки в темном поле D. Staiger и С. Hemtzen)

A—tissue of an onion root tip (Allium cepa) after a ³H-thymidine pulse-label. Nuclei whose DNA replicated during the pulse (S-phase), following photographic emulsion development performed over the section, are packed with numerous black silver grains. Unlabeled nuclei were not in the S-phase during radioisotope Treatment. DNA-free cellular structures were not labeled with ³H-thymidine (380x). B–E—detection of transcripts (mRNA) by in situ Hybridization with synthetic radioactive RNA probes on cross sections of white mustard shoots (Sinapis alba). B—with vascular bundles L, cortical tissue R, and cambium, arrow. C, D—differential transcriptional activity of a gene encoding one of the RNA-binding proteins depending on the time of day (C—maximum at the end of the photoperiod, cambium binds intensively to the labeled RNA probe; D—minimum, no label detected; lignified parts of vascular bundles autofluoresce in dark-field without being labeled). E—mRNA for The Cell wall protein is produced exclusively in the outer cortical cells (60x).

Various fluorescent methods Complement microradioautography, a technique that was frequently used in the past. This extremely sensitive method is based on the specific incorporation of radioactive isotopes into particular substances or structures of living cells (for example, tritium-labeled thymidine into DNA, ³H-uridine into RNA, or 30S-Methionine into proteins). Following appropriate prolonged dark exposure and development, silver grains form on the photographic emulsion that covers thin sections of labeled cells or tissues directly above the radionuclide-containing structures of the specimen (Fig. 2.4).

In many studies, it is crucial to purposefully manipulate individual cells. To this end, expensive micromanipulators are available, and recently, laser systems ("optical tweezers") have increasingly replaced mechanical devices.

Alongside novel light Microscopy Techniques, a range of other modern cell research methods plays a vital role. These are often based on growing genetically homogeneous cell clones in cell culture (see Fig. 2.1). Protoplasts, stripped of their walls through enzymatic treatment, enable the application of numerous methods originally developed for animal and human cells. These include artificial Cell Fusion (see Fig. 2.49) and patch-clamp techniques for investigating Ion Channels and receptors. To purposefully manipulate individual cells, The cell membrane must become permeable—at least temporarily and/or locally—allowing experimental modulation of various metabolic parameters (such as ionic environment, pH value, energy level, etc.). Specially prepared permeabilized cells¹ (whose cell membrane is rendered permeable by detergents) or so-called cell models (partially active cell remnants lacking a cell membrane, which are short-lived) serve this purpose. Microinjection offers an alternative approach. Macromolecules can also be introduced into living cells via electroporation (creating permeable patches in the cell membrane using brief electrical pulses) and ballistic methods, or biolistics (DNA- or RNA-loaded gold or tungsten particles approximately 1 εm in diameter are accelerated via a Shock wave into, for example, leaf tissue)². Such methods make it possible, for instance, to specifically block certain Enzymes in living cells using introduced antibodies, or to artificially alter genetic activity through the targeted introduction of foreign DNA (transfection), Transcription factors, or antisense mRNA.

¹ From the English "permeable." This term is rarely used in domestic literature. — Ed. note.

² Originally, biolistics utilized gunpowder-loaded cartridges and fired from a gun (a so-called "gene gun"). Today, gentler methods for generating shock waves using compressed gases or a solenoid have been developed, which accelerate metal particles via magnetic forces. — Ed. note.

— Ed. note.

2.1.2. Electron microscopy

In the electron microscope (EM, Fig. 2.5), specimen illumination and imaging are performed by rapid electron streams refracted by electromagnetic lens fields. The magnified image is projected onto a fluorescent viewing screen and can be captured as photographs or digital data. Following acceleration at 100,000 V (= 100 kV), the wavelength of the electron beams is merely 1/100,000 of the wavelength of light. Consequently, a much higher resolution is achieved compared to light microscopy. For biological specimens, resolution is enhanced by two orders of magnitude, which is of paramount importance.

For analysis using a conventional transmission EM (TEM), specimens must be no thicker than 80 nm—less than 1/1,000 of the thickness of a sheet of paper. Several preparation methods exist for TEM. Transmitted particles (macromolecules, multienzyme complexes, DNA strands, Ribosomes, Viruses, Cellulose fibrils, membrane fractions) are dried on ultrathin plastic or carbon films and observed directly. To enhance image contrast, heavy metals are often applied by negative staining (positive contrast), surface deposition (negative contrast; cf., e.g., Figs. 1.16 A, 1.17, 2.44, 2.66, 2.80), or oblique shadowing (relief-effect shadowing, Fig. 2.72). Following chemical fixation with glutaraldehyde and osmium tetroxide, Cells and Tissues are embedded in a hard polymerizing resin and sectioned on ultramicrotomes using specially sharpened diamond blades (cf., e.g., Figs. 2.7, 2.93). Alternatively, cryofixation can be employed via rapid freezing of living tissue down to <-150°C, preventing ice crystal formation. The frozen specimen is then fractured, and a thin replica of the fracture surface is prepared for TEM observation (freeze-fracturing, cf., e.g., Figs. 2.18, 2.26 A, 2.85, 2.94 A, C). Recently, relatively thick sections have also been examined using accelerating voltages between 300 and 700 kV, yielding digital images of corresponding specimen areas. Using computer processing, these data are used to reconstruct a virtual three-dimensional rendering of the object that reflects its Spatial Structure (much like a confocal laser scanning microscope).

Fig. 2.5. Modern electron microscope.

Electron beams travel from source 1 from top to bottom through a condenser lens system within the Column (vertical tube 5), reach the specimen placed in the high-vacuum region of the column (specimen airlock 2 with side Dewar flask 4 for liquid nitrogen to cool the specimen chamber; 3 — specimen-tilting device), pass through the fields of the electromagnetic objective and projector lenses (at 5), and finally strike the fluorescent viewing screen. The resulting final image can be observed through the viewing window (6) or on monitors (8), and photographed or saved digitally (digital camera 7). Residual gas pressure in the column is maintained by vacuum pumps at one-millionth of an atmosphere. 9 — computer unit for image acquisition and processing.

The surface structure of opaque objects can be visualized using scanning electron microscopy (SEM). This method operates on THE PRINCIPLE OF television technology. A highly focused electron beam scans a restricted area of the specimen surface. Secondary electrons¹ are backscattered from the specific points on the specimen directly struck by this primary beam. These electrons synchronously control the digitization of the surface image on a monitor screen. There are no imaging lenses. SEM images are characterized by a high depth of field and a particularly vivid, three-dimensional representation of the object's topography (cf., e.g., Figs. 3.3 C, D; 3.10, 3.11, 3.14).

¹ Before examination, a thin metal film (e.g., palladium) is sputtered onto the specimen, because living specimens themselves transmit electron beams well and reflect them poorly — Ed. note.