Eukaryotes

Eukaryotes: Membranes, Cytoskeleton, Organelles, and Key Concepts

  • Overview: Eukaryotic cells are more complex than prokaryotes due to extensive internal membrane systems and a sophisticated cytoskeleton. In contrast to bacteria, which have only the plasma membrane, eukaryotes contain membranes around organelles such as chloroplasts, mitochondria, Golgi, endoplasmic reticulum, and the nucleus.

Plasma membrane and membrane sidedness

  • Plasma membrane is a phospholipid bilayer with a hydrophobic core formed by fatty acid tails.

  • Integral proteins span the membrane and allow ions and molecules to cross.

  • Inside surface of the plasma membrane is labeled by actin (microfilament) to maintain cytoskeletal shape.

  • Outside surface toward environment contains carbohydrate branches (glycocalyx).

  • Sidedness: actin is on the inside; carbohydrate branches are on the outside.

Cytoskeleton and endocytosis (three main endocytic pathways)

  • Eukaryotes have a complex cytoskeleton with three components:

    • Microtubules (largest)

    • Intermediate filaments (medium)

    • Microfilaments/actin (smallest)

  • Prokaryotes lack this complex system (at most actin homologs like acanthin).

  • Endocytosis (bringing material into the cell):

    • Phagocytosis: membrane extends to surround bulk particles, engulfs them, vesicles form and internalize into the cell. Used by immune cells to engulf bacteria (e.g., phagocytosis of E. coli).

    • Pinocytosis (pentocytosis in some slides): membrane invaginates and forms a vesicle to bring in extracellular fluid and solutes indiscriminately.

    • Receptor-mediated endocytosis: similar to pinocytosis but selective, bringing in substances that bind to specific receptors.

  • Exocytosis (export): vesicles fuse with the plasma membrane and release their contents to the outside.

  • Note: All three endocytic processes are performed by eukaryotes; prokaryotes do not perform these in the same way due to the lack of a complex cytoskeleton.

Cytoskeletal architecture and function

  • Microtubules: hollow tubes that can act as railroad tracks for moving organelles within the cell.

  • Intermediate filaments: form a supportive web (spider-web network) that helps maintain structure.

  • Actin filaments: lie just inside the plasma membrane and aid in movement and stability.

  • Motors and trafficking rely on coordination of all three components.

  • Flagella and cilia are built from long and short microtubules, respectively, and move the cell or propel extracellular movement.

Flagella: prokaryotes vs eukaryotes

  • Both serve to move the cell, but are structurally and functionally different and should not be confused purely by name:

    • Prokaryotic flagella: extracellular, powered by a proton gradient (proton motive force), made of flagellin. Movement style differs from eukaryotes.

    • Eukaryotic flagella: intracellular and covered by the cytoplasmic (plasma) membrane; movment is driven differently and their structure is based on microtubules.

  • Important difference highlighted: In prokaryotes, flagella are typically extracellular; in eukaryotes, flagella are intracellular and surrounded by the plasma membrane.

  • Spirochetes are an exception discussed: they are intracellular in the context of this lecture, which contrasts with the typical prokaryotic extracellular flagella.

  • Energy source for movement:

    • Prokaryotes: energy from the proton gradient (proton motive force).

    • Eukaryotes: energy directly from ATP.

Ribosomes: prokaryotes vs eukaryotes

  • All cells have ribosomes for protein synthesis, but composition differs:

    • Prokaryotes: large subunit composed of 34 ribosomal proteins with two rRNA species.

    • Eukaryotes: large subunit composed of 49 ribosomal proteins with three rRNA species.

  • Both have a small subunit and carry out protein synthesis to build proteins.

  • Implications: Some antibiotics target bacterial ribosomes; selectivity is based on differences from human ribosomes, which is a therapeutic consideration discussed later in the course.

Nucleus and DNA organization

  • Prokaryotes lack a true nucleus; eukaryotes have a nucleus enclosed by a nuclear membrane.

  • DNA in the nucleus is linear and packaged with histone proteins into chromatin.

  • Beads-on-a-string structure: DNA wraps around histones (eight histones form a core octamer; the DNA wraps around twice to form a bead-like unit).

  • The nucleolus: a dark-staining body inside the nucleus where ribosomal RNA (rRNA) is synthesized; more rRNA is produced than any other RNA because ribosomes are needed in high numbers to make proteins.

Mitochondria and chloroplasts: energy producers with their own genomes

  • Chloroplasts: site of photosynthesis in plants and algae; convert energy from the sun into chemical energy (sugar).

  • Mitochondria: powerhouse of the cell; generate ATP.

  • Both organelles have their own DNA and ribosomes, separate from nuclear DNA and cytoplasmic ribosomes.

  • DNA organization: mitochondrial and chloroplast DNA are circular, similar to bacterial DNA; nuclear DNA in humans is linear; chloroplast DNA is circular; mitochondrial DNA is circular; plants and some other organisms may have additional DNA forms.

  • Endosymbiotic theory: proposes that chloroplasts and mitochondria originated as free-living bacteria that were engulfed by a primitive eukaryotic cell and eventually became organelles.

  • Implications for drug action: some antibiotics target bacterial ribosomes; such drugs can affect mitochondrial ribosomes and potentially disrupt energy production, highlighting the importance of selective targeting.

  • Practical note: in trees, three DNA types exist within a single cell (nuclear linear DNA, mitochondrial circular DNA, chloroplast circular DNA).

Endoplasmic reticulum (ER) and Golgi apparatus

  • The ER is formed from the outer nuclear membrane folded into the cytoplasm.

  • Two distinct regions:

    • Rough Endoplasmic Reticulum (RER): has ribosomes on the cytoplasmic side; functions to fold and modify proteins that are secreted, membrane-bound, or digestive in nature.

    • Smooth Endoplasmic Reticulum (SER): lacks ribosomes; functions include:

    • Calcium storage and release to regulate protein activity (calcium signaling).

    • Lipid synthesis (phospholipids, cholesterol), and other lipid-related products.

    • Minor detoxification roles, making some compounds water-soluble for secretion.

  • Protein folding and processing:

    • Many proteins are not functional upon synthesis; they require folding and modification to become active.

    • Proteins destined for secretion, membrane integration, or lysosomal targeting pass through the ER, where folding begins.

  • Golgi apparatus: processes and further folds/modifies proteins after the ER; organized as a stack of distinct cisternal layers, with a directional flow from the ER toward the plasma membrane.

    • Proteins leave the Golgi in vesicles; destination dependent on the signal:

    • Secreted proteins are packaged into vesicles that fuse with the plasma membrane and release their contents outside the cell.

    • Digestive enzymes are packaged for lysosomes and become functional there.

  • Lysosomes: acidic organelles where intracellular digestion occurs; digestive proteins become fully active in lysosomes.

  • Additional organelles:

    • Peroxisomes: break down hydrogen peroxide and degrade D-amino acids (humans primarily use L-amino acids; D-amino acids are removed by peroxisomes).

Laboratory concepts: pure culture, asepsis, and culture techniques

  • Pure culture: a population descended from a single cell; contains only one bacterial type.

  • Mixed culture: contains several different bacteria (multiple species) in the same tube.

  • Plate cultures and colonies:

    • A colony represents a clonal expansion from a single bacterium that has divided many times to form visible growth.

  • Sterile conditions and aseptic technique:

    • Learn to manipulate tubes and equipment to avoid contaminating samples with other microbes.

    • Bacteria are ubiquitous; aseptic technique is essential to prevent contamination.

  • Agar plates: growth medium made from polysaccharide agar that forms a solid surface for plating and colony development.

  • Sterilization and prions:

    • Sterile typically means free from bacteria, but the meaning is context-dependent and should specify what is being removed (bacteria, fungi, viruses, prions).

    • A real-world example (2013, New Hampshire): hospital equipment sterilized according to manufacturers’ instructions was free of bacteria and viruses but not prions, leading to Creutzfeldt-Jakob syndrome.

    • Prions require specialized, extended autoclave conditions (e.g., about five hours at high temperature) to effectively inactivate.

    • Regular autoclaving (20 minutes) kills bacteria but does not inactivate prions; long processing times would reduce throughput.

  • Practical takeaway: always specify what is meant by sterile and what pathogens or agents are being targeted; prions require different sterilization protocols than bacteria/viruses.

Practical lab technique: streak plating to obtain pure cultures

  • Midterm/practicum task: given a tube of mixed cultures, separate individual bacteria into isolated units on an agar plate using streaking:

    • Use a sterile loop to streak from the original tube onto the agar plate, then re-sterilize the loop and streak through progressively cleaner quadrants (quad 1 to quad 4).

    • The goal is to reduce bacterial density in each quadrant; eventually, individual colonies appear in the later quadrants.

    • Rule of thumb for grading: at least five isolated, non-touching colonies are needed for a portion of the grade.

  • This process converts a mixed culture into a pure culture by isolating single colonies.

  • Important note: not all ribosomes are attached to the ER; many ribosomes are free in the cytoplasm and synthesize non-secreted proteins.

Bacterial growth: binary fission and doubling time

  • Bacteria reproduce by binary fission (not mitosis/meiosis): a single cell grows large and then splits into two.

  • Doubling time: the time required for a bacterial population to double in size; varies by species.

  • Example: E. coli doubles every 20 minutes.

  • Growth calculation example:

    • Suppose 10 bacteria contaminate potato salad (N0 = 10).

    • If the potato salad sits for 4 hours (t = 4 hours) and E. coli doubles every 20 minutes, the number of doubling periods is:

    • There are 12 twenty-minute periods in four hours (
      4 ext{ hours} = 240 ext{ minutes}; ext{ 240}/20 = 12.)

    • The population after four hours is:
      Nt = N0 imes 2^{n} = 10 imes 2^{12} = 10 imes 4096 = 40{,}960.

  • Consequence: consuming contaminated potato salad after this period would likely cause illness due to high bacterial load.

  • Other doubling times: Staphylococcus ~20 minutes; Mycobacterium tuberculosis can take up to a day; Mycobacterium leprae can take about ten days.

  • Note on selecting doubling time: E. coli is commonly used in the lab due to rapid division.

Quick exam/quiz context

  • The class will have an open-notes quiz covering the material above.

  • A practical lab exercise will be conducted to reinforce culture isolation techniques, endocytosis concepts, organelle functions, and the differences between prokaryotic and eukaryotic cells.