Cell Structure and Microscopy

Introduction to Cells

• Cells constitute the fundamental units of life and the building blocks of living organisms
• Cell principles include the following:
  • Cells are fundamental units of life
  • All living organisms consist of cells
  • Existing cells divide to create all new cells
• The study of cells approximates the study of life
• Life is continuous; all cells derive from a single fertilized egg

Microscopes

• Microscopes show key cell features
• Magnification increases the apparent object size by magnifying it's real size
• Resolution provides clarity and prevents pixelation with higher resolutions
• Contrast illuminates variations in brightness across sample sections
• Cellular structures are observable when all these factors function together

Cell Size

• Cell size widely varies between a 1-100 μm diameter
• Large cell structures can be seen without assistance, for example; nerve and egg cells
• Microscopy is crucial when studying smaller structures
  • Light microscopy is used on most plant and animal cells plus nuclei and most bacteria
  • Electron microscopy is used on smaller bacteria, viruses, ribosomes, and proteins
• Technology is relied upon to understand cell structure and biological concepts

Light Microscopy Types

• Light passes directly through cells in brightfield microscopy without staining so contrast and detail is minimal
• Brightfield microscopy enhances contrast of stained specimens allowing the observation of the nucleus, and plasma membrane
• Phase-contrast highlights lightly and darkly staining-free regions within the cell
• Nucleus and membrane structures are viewable, however, internal details are not
• Differential Interference uses two beams of polarized light to create shadow effects for cell structure visibility

Advanced Light Microscopy

• Molecules can be labeled inside cells when utilizing fluorescent dyes or antibodies during fluorescence
• Fluorescence detection occurs with the absorption of ultraviolet (UV) light, and the emission of visible light
• DNA, mitochondria, and the cytoskeleton can be highlighted
• Confocal microscopy focuses a laser on a single optical plane that eliminates out-of-focus light
• Multiple layers can be imaged to then create a 3D reconstruction
• Deconvolution applies software to remove light that is out-of-focus
• Fluorescence images have sharpness and resolution improved
• Confocal microscopy can be enhanced with 3D visualisation

Electron Microscopy (EM)

• Electron microscopes focus electron beams, rather than light using electromagnets, which then enables much higher resolution
• A vacuum is required, and the sample gets placed in an airless chamber for imaging
• Living cells cannot survive and must be fixed and preserved beforehand
• Scanning Electron Microscopy (SEM) detects released electrons and produces a detailed surface image
• Transmission Electron Microscopy (TEM) detects passing electrons to produces high resolution internal structural images

Light Microscopy Methods

• Brightfield (unstained) uses light that passes directly through the specimen providing minimal contrast, for the viewing of living cells with minimal contrasting detail
• Brightfield (stained) enhances contrast with specific wavelengths of light to provide enhanced visibility of the structures, for example the nuclei
• Phase contrast uses light phase shifts to enhance transparent specimen contrast for observing live cells with no staining
• Differential Interference Contrast uses polarized light to enhance transparent specimen contrast and improve visuals with 3D-like effect
• The use of fluorescent dyes or proteins to label specific structures and excite them with UV light allows the identification of molecules
• Confocal Microscopy uses lasers focused on a single plane to eliminate out of focus light and create generating high-resolution images of the cell sections and assist with 3D reconstruction
• Deconvolution uses a computational method to remove out of focus light and sharpen images, enhancing fluorescence images digitally
• Scanning Electron Microscopy detects scattered electrons from the specimen to create a 3D surface representation, for the study of cell surfaces and morphology
• Transmission Electron Microscopy passes the electron through an ultrathin specimen to reveal high resolution internal structural details, for examination of internal cell structure

Cell Fractionation

• Cell fractionation separates cell components by size and density
• The steps include:
  • Homogenization: Breaking apart cells using blending, grinding, or lysing to release the the contents by disrupting the plasma membrane
  • Homogenate Formation: The resulting suspension of cell fragments and organelles
  • Centrifugation: The homogenate is spun at a high velocity to separate components by density and their shape

Differential Centrifugation

• Organelles get separated via centrifugation in steps that progressively isolate smaller components
• The first centrifugation step involves:
  • Pellet formation at the bottom by the heaviest components (nuclei and cellular debris)
  • Supernatant transfer to a separate new tube
• Centrifugation is repeated at progressively higher speeds, separating out smaller and lighter organelles each time
• The smallest components, namely ribosomes, get isolated last during the final spins requiring highest speeds
• Researchers can select the speed and time to isolate specific organelles for testing

Prokaryotic cell anatomy

• Prokaryotic means “before nucleus” → lack a membrane-bound nucleus
• The nucleoid contains DNA composed of a single circular molecule in a concentrated region
• Key structures in prokaryotic cells:
  • Plasma membrane facilitates entry and exit of substances
  • Cytosol facilitates cellular processes in fluid
  • Ribosomes synthesize proteins
• Presence of organelles do not delineate this a definition for this cell-type because some species cells include them
• Many prokaryotic cells travel using flagella
• Almost all cells also have a cell wall that helps maintains shape
• Prokaryotic cells exhibit an elemental internal organization and compartmentalization when contrasted with eukaryotic cells

Eukaryotic vs Prokaryotic

• Eukaryotic cells utilize membranes to compartmentalize functions for efficient cellular operations
• Distinct differences when compared with to prokaryotic cells:
  • Larger in size when compared to prokaryotic cells
  • A nucleus encloses chromosomes; while there is a nucleoid, which chromosomes are loosely dispersed within the cell
  • Cytoplasm is compartmentalized via membrane-bound organelles, while components are dispersed in prokaryotic cells
• Compartmentalization advantages include:
  • Prevents reactions from occurring in the same cell space by utilizing separate areas
  • Enzymes are grouped together that effectively catalyze reactions, improving efficiency by maintaining a higher concentration of reactants

Plasma Membrane Makeup

• Found in all cell-types the plasma membrane is essential
• The membrane functions as a selective barrier that controls the entry of important molecules and prevents harmful substances from the cell
• Its structure consists of:
  • Phospholipids - arrange into a bilayer with a hydrophilic head and a hydrophobic tail
  • Proteins - are either embedded or attached; assist with signalling, transport and structural function
  • Carbohydrates - aid in cell recognition and are linked to proteins and lipids
• Scale and Importance:
  • Compartmentalization is aided by the thin-structured membrane
  • Internal eukaryotic membranes aid in maintaining specialized environments

Cell Size and Area

• The limited size is due to the need for nutrient and waste exchange
• As cells increase in size volume grows faster than the surface area
  • Surface area increases as a square function (r²)
  • Volume increases as a cubic function (r³)
• Results in less available membrane to exchange relative to requirements
• Larger cells have a lesser surface area-to-volume ratio, limiting exchange
• Efficiency exchange becomes more efficient when cells decrease in size which impacts on the higher surface area
• When a cell is broken into smaller units, the total surface area is increased
• Surface area expands due to specialized cells folds or projections
  • Microvilli enhance nutrient absorption by increasing surface area without increasing volume

Nucleus and Ribosomes

• Replication and transcription of DNA occurs with chromosomes within the nucleus
• A double membrane creates an envelope around the nucleus acting as a protective barrier
• RNA and proteins move in and out of the nuclear envelope through its pores
• Proteins forming the nuclear pore complex (NPC) control import and export of nucleus substances
• Nucleoli:
  • Specific regions within the nucleus
  • Generate and assemble ribosomal components from transcribed ribosomal RNA (rRNA)
• Instructions are assembled by ribosomes

Ribosomes

• Macromolecular assemblies composed of RNA and proteins
• Responsible for polypeptide formation in protein synthesis
• Classic definition excludes these from being organelles due to the absence of a membrane enclosure
• Ribosomes exist in two forms:
  • Proteins are made by free ribosomes that either stay in the cytoplasm or get transported to organelles
  • Bound ribosomes produce proteins that follow an pathway in the endomembrane system
• Protein synthesis involves both subunits assembled
• Held together with non-covalent bonds to assemble and disassemble quickly

Endomembrane System

• Endomembrane dictates the actions of metabolic functions and moves proteins
• Components include:
  • Nuclear envelope
  • Endoplasmic reticulum (ER)
  • Golgi apparatus
  • Lysosomes
  • Vacuoles & vesicles
  • Plasma membrane
• Vesicle transport dictates interactions and continuity between these parts
• Secretion and protein-to-location placement and direction is dictated by the system

Endoplasmic Reticulum

• Membrane bound organelle continuous with the nuclear envelope
• This organelle comes in two distinct types:
  • Smooth ER (SER):
    • Enzymes aid with breakdown and lipid synthesis
    • Production of phospholipids for cell membranes
    • Toxic substances and harmful molecules are removed through detoxification
    • Calcium storage for cellular signaling
  • Rough ER (RER):
    • Ribosome-derived rough appearance
    • Polypeptides include
    • Those that continue to remain in the ER
    • Those that move through the endomembrane system
    • Those that are secreted externally to the cell
    • Protein folding and modification occur in the ER lumen
    • Proteins get glycosylated for folding support and quality control
• SER to RER ratios are dependent on the cells function - Lipids: SER has more - Proteins: RER has more

Golgi-Apparatus

• The main function includes protein processing from the rough ER that are then directed, sorted, and modified
• Flattened sacs called cisternae form its structure that stack on top of one another
• New proteins are received from vesicles close to the endoplasmic reticulum at the cis face
• Modifications occur as proteins traverse the cis to the trans face that include glycosylation and/or the addition of molecular tags
• Vesicles package final protein products and distribute, at the trans face - Placement into various other organelles, lysosomes - Insertion into plasma membrane - Return to the ER
• The mechanisms by which proteins pass through the Golgi vary

Lysosomes

• Play a critical role in recycling and cell-digestion
• Hydrolytic enzymes breakdown proteins, lipids, carbs, and acids via hydrolysis, in to building blocks
  • Hydrolases are most active in high level acidic spaces
• It maintains its high acidity with proton pumps that actively transport H+ ions into its lumen
• Macromolecules get digested for raw molecules to build other molecules
• In addition, lysosomes also function as waste disposal and recycle to assist in maintenance
• Starting at the ER and processed in the Golgi; membrane and proteins end up here as a crucial part of the endomembrane system

Phagocytosis: A Lysosome Function

• Engulfment and enclosing of large particles or microorganisms within a membrane is termed cellular phagocytosis
• Forms a food vacuole called a phagosome after the plasma membrane surrounds material
• Phagosome fuses with the lysosome that contains the digestive enzyme and also comprises of acid hydrolases
• Allows cells to extract and or expel from useful substances, the enzymes breakup material inside
• Both single celled and large organisms such as white blood cells engulfing harmful pathogens perform phagocytosis and aid in immune defense

Autophagy

• In this recylinc process damaged, age cell parts are rebuilt with new components
• Portions of inner cytoplasm or aged organelles get enclosed by the formation of an internal autophagy membrane
• Degradative enzyme containing lysosomes will fuse with digested autophagy
• It facilitates recycling of components, where it discharges back into cytosol
• Assists with starvation when nutrients materials can be recycled to self-recycle its efficiently

Vacuoles

• Plant vacuole functions consist of replacement for lysosomes

• Constitutes bulk in cell size

• Consist of hydrolytic enzymes; stores

• The various types are:

  • Color
  • Energy source
  • Safety measure

• Important for cell structure even though dimensions change through plants

Endomembrane Summary

• Nucleus
• Nuclear envelope
• Smooth ER
• Rough ER
• cis Golgi
• trans Golgi
• Plasma membrane

Endomembrane Transport

• Endomembrane utilizes proteins that reach correct locations through a number of steps
• Protein synthesis begins in the rough ER
  • The nuclear envelope connects directly to the ER to make a continuous membrane
  • Synthesis is performed on the rough ER
• Golgi apparatus processing
  • Golgi cis phase vesicles transport
  • Proteins get modified through tranz transition
• Final Destination
  • Vesicles bud from the trans Golgi and transfer proteins across
    • Lysosomes and vacuoles digest cells via digestion for storage
    • Membranes are created by secretion in the plasma membrane
    • The proteins are transported where needed
• After completion proteins fuse for secretion - Exocytosis releases content

Role of Mitochondria

• Essential in metabolism when working its energy creation
• Respiration is performed through membranes for efficiency
• In coordination with internal and external boundaries, it moves chemicals
• Folds that create surface increasing are referred to as cristae
• Key synthesis occurs between membranes
• Key reactions occur in the matrix to carry out metabolic activity
• Higher concentrations found when more energy is expected

Mitochondrial DNA

• These mitochondria have a different DNA that isolates themselves from main genetic material
• Genetic material is circular, and not linear
• A portion is assigned to this mitochondria
• RNA for the genetic formation is used for proteins
• Protein manufacturing and cytoplasm introduction are done by ribosomes

Chloroplasts

• Chloroplasts convert light and help the photosynthetic process that provides energy
• Chlorophyll is extracted and enzymes are used for photosynthesis
• Greens on pants mostly and algae are comprised of leaves
• Analogous membrane in the structure to mitochondria; thylakoids
  • Inner membranes are stacked in grana
• These conversions are used efficiently

Endosymbiotic Theory

• Bacteria engulfed by cells are termed mitochondria/chloroplasts
• Support factors encompass - Double membranes - Reproduction functions independently
• Eukaryotic cell also engulfs photosynthetic cells
• Useful relationships give rise