BIOMG 1350 Lecture 1 - Cells and Organelles

3 domains of life and their common origin

Bacteria, Archaea, Eucaryote

All life has common origin 3 billion years ago

Two empires of life

Prokaryotes: Eukarya and Prokarya

Eukaryotes: Eukarya

How do we know about the relatedness between organisms?

DNA sequences of all organisms are related

The basic chemistry of all cells is similar

The fundamental processes of organisms and their cells are similar ("are evolutionary conserved")

Implication of these facts: What you learn about one type of cell will likely be true in other cell types.

Prokaryotes (Bacteria and archaea)

Single celled organisms (have not evolved multicellularity) without a nucleus

No membrane bound organelles and very diverse (more diverse than Eukaryotes)

Evolutionarily very successful

Are all unicellular

Eukaryotes (eukarya)

They have specialized subfractions or organelles that can perform specific functions and is partly why they can form multicellular organisms.

Contain nuclear membrane and membrane bound organelles.

Idealized version of Eukaryotic cells contents: true nucleus, nuclear envelope, lysosome, mitochondrion, peroxisome, plasma membrane, Golgi apparatus, endoplasmic reticulum (rough and smooth), and vesicles.

Formed via endosymbiosis

“eu” means truly and “karyon” means nucleus in greek

Endosymbiosis

Endosymbiosis is a key step that allowed eukaryotes to be created

We think an early prokaryote (archaea) embedded itself into another archaea cell (specifically a smaller prokaryote embedded itself which could produce oxygen and it turned the once archaea cell into the modern eukaryotic cell) the basis of a eukaryote with organelles is formed

Endosymbiosis only happened once as it doesn’t really happen again and thus scientists think its exceedingly rare

The ancestor or plant cells probably took up another prokaryote that eventually turned into chloroplasts

This event occured around 1.6 to 2.2 billion years ago

Why do eukaryotic cells contain internal membranes?

• Allows for compartmentalization of cellular functions

  • Cells perform many competing reactions that need to be separated

• Allows for more membrane surface per cell volume

  • Many more reactions can be carried out in membranes

  • Larger cells have a smaller ratio of cell surface to volume

How do we investigate cells and their inner structure

Via microscopy

3 different major types: Light microscopy, fluorescence microscopy, and electron microscopy

Light microscopy

Visible light has wavelength of 400 to 700 nm

Since light microscopes use visible light they can resolve objects at or above 200nm apart

Uses visible light and glass lenses to magnify specimens and form an image

Viewing whole cells, tissues, and larger structures; can view living cells.

Resolving Power

Resolving power is the ability to distinguish two close objects. Light microscopy cannot distinguish two dots 100nm apart whereas an electron microscope can

Maximum resolving power depends on the wavelength of the illumination

Light microscopy has a wavelength of 400 to 700nm and can resolve objects 200nm apart

In a 200kV electron microscope the wavelength of the electrons is 3 picometers (0.003 nm) and can resolve atoms (around 0.2 nm resolving power).

LUCA

Last universal common ancestor of eukarya, prokarya, archaea

Size Scales Relevant to Biology

1m = 10³ mm (milimeter)

1m = 10^6 μm (micrometer)

1m = 10^9 nm (nanometer)

or

1 mm = 10^-3 m

1 μm = 10^-6 m

1 nm = 10^-9 m

Electron microscopy

Wavelength of electrons is 3 picometers and this allows the electron microscope to resolve atoms, ribosomes, and cell membranes in very fine detail

Uses a beam of electrons instead of light to produce highly magnified, high-resolution images.

Viewing very small cellular structures that light microscopes cannot resolve, molecules, or atoms

0.2 nm is the minimum resolvable distance by electron microscopes (200 picometers)

Two types: Transmission Electron Microscopy (uses electrons that pass through a very thin specimen to produce a detailed image of its internal structures) and Scanning Electron Microscopy (uses electrons to scan the surface of a specimen, producing a detailed image of its surface structure)

Fluorescence microscopy

Since fluorescence microscopes use visible light as well they can resolve objects at or above 200nm apart

Uses fluorescent molecules (fluorophores) that emit light after being excited by specific wavelengths, allowing specific structures to be visualized

Locate specific molecules/structures inside cells; can label proteins, organelles, DNA, etc.

Minimum resolvable distance by unaided eye

0.2 mm (200 micrometers)

basically your eye is not a good microscope but its good for other things

Cells have thousands of different proteins. How do we visualize a protein of interest?

Human cells contain about 20,000 different proteins. Cells have thousands of different proteins. How do we visualize a protein of interest?

The abundance of each type of protein is different, some may be present in a few hundred copies, whereas others may be present in many million copies.

We can study or visualize an individual protein despite there being a lot of them through florescence. A molecule is fluorescent if it can absorb light of one wavelength and then emits slight of a longer wavelength.  

• We can harness the green fluorescent protein (GFP) which is fluorescent and in nature

• We can add the GFP gene to the genome right next to the gene of the protein of interest, and then when the protein of interest and GFP gene is transcribed then translated, the protein is attached to the fluorescent gene allowing scientists to follow proteins

Fusing proteins to green fluorescent protein (GFP) transformed cell biological studies, allowing for the localization of specific proteins in living cells by fluorescence microscopy.

Green Fluorescent Protein (GFP)

We can harness the green fluorescent protein (GFP) which is fluorescent and in nature

We can add the GFP gene to the genome right next to the gene of the protein of interest, and then when the protein of interest and GFP gene is transcribed then translated, the protein is attached to the fluorescent gene allowing scientists to follow proteins 

Different types of similar proteins including GFP for different colors (so we can color code): RFP, BFP (red and blue)

CFP (Cyan) and YFP (yellow) was made by muytating key amino acids in GFP to make CFP and YFP

From the jellyfish aequoria victoria

Fluorescence

A molecule is florescent if

1) it absorbs light of one wavelength and then

2) emits light of a longer wavelength

(this is a very sensitive process)

The molecule can absorb one wavelength and a bit of its energy is used to excite electrons and then a less energetic wavelength is emitted

ex: UV/blue light is shone on a molecule, green light (of a longer wavelength once energy is released) is emitted making the sample fluoresce green

Plasma membrane

Separates the cell from the environment and mediates interactions with the environment (signaling, nutrient uptake, endo and exocytosis)

Cytoplasm

Everything in between the plasma membrane and the nucleus (including organelles)

Cytosol

The soluble portion of the cytoplasm outside of organelles and they contain many RNAs, proteins, ribosomes, many chemical reactions including protein synthesis occur though large no organelles within it.

It accounts for about 50% of the volume of a typical cell. Many chemical reactions occur in the cytosol, including most of the protein synthesis.

Nucleus

Contains the genome (most of cellular DNA). Replication and transcription occur in the nucleus

Darker regions of the nucleus are the nucleolus where the ribosomes are assembled

Nucleus is surrounded by a double membrane (inner and outer) and this allows for an exchange of material via nuclear pores

Endoplasmic reticulum (ER)

ER is contiguous with the membranes that surround the nucleus

Primary site of synthesis of lipids, membrane proteins, and secreted proteins

• Rough ER: Covered in ribosomes and primarily makes, folds, and modifies proteins that will be secreted, inserted into membranes, or sent to certain organelles.

• Smooth ER: Lacks ribosomes and is mainly involved in lipid synthesis, detoxification, and Ca²⁺ storage.

Golgi Apparatus

Modification of secretory proteins, akin to postal station

Sorting station of vesicle trafficking

Contains membrane enclosed vesicles stacked like multiple pancakes

The Mitochondria

Contain an outer and inner membrane, and the inner membrane is folded with cristae. They are not Beans!

Major site of ATP production (oxidative phosphorylation) and produces central metabolites (amino acids and nucleotides)

Evolved from engulfed bacteria and still contain their own DNA that encodes some of their proteins and RNAs

Lysosome

Digestion and recycling. Contain digestive enzymes that break down various things in the cell. Breaks down old or damaged cell parts, cellular waste, or material brought into the cell.

Peroxisome

Oxidation and detox. Oxidation reactions occur here and harmful substances are detoxified. Produces hydrogen peroxide in some reactions and uses catalase to convert H2O2 into water + O2.

Size Scales in biology and what they are used to Measure

Unit	Size	Think of...	Biology examples
Meter (m)	10⁰ m	Whole organisms / large structures	Humans, trees, large animals
Centimeter (cm)	10⁻² m	Body parts / larger structures	Organs, leaves, insects
Millimeter (mm)	10⁻³ m	Small visible structures	Small organisms, embryos, tissues
Micrometer (μm)	10⁻⁶ m	Cells & cellular structures	Most animal/plant cells, bacteria, mitochondria
Nanometer (nm)	10⁻⁹ m	Molecules & very small structures	Viruses, ribosomes, proteins, DNA width, cell membrane thickness
Picometer (pm)	10⁻¹² m	Atoms & chemical bonds	Atoms, atomic radii, bond lengths

What are membraneless organelles?

Structures within cells that lack a surrounding lipid membrane but organize specific molecules and carry out specialized functions. They often form through phase separation of proteins and RNA.

Examples: nucleolus, stress granules, P-bodies.

When were cells discovered

17th century. The invention of the microscope (light) lled to the discovery of cells in this year.

Cell and Organelle Sizes (Largest to Smallest)

Cell: ~10,000–30,000 nm (10-30μm)

↓

Bacteria: 1,000 nm (1μm)
↓
Nucleus: ~5,000–10,000 nm (5 to 10 μm)
↓
Mitochondrion: ~500–10,000 nm (0.5 to 10 μm)

↓

Typical Organelle: 1,000–2,000 nm (1 to 2 μm)

↓
Ribosome: ~20–30 nm
↓
Protein: ~2–10 nm
↓
Small molecule: ~1 nm
↓
Hydrogen Atom: ~0.1 nm

Cell Theory

1) all living organisms are made of cells

2) cells are the basic unit of life

3) cells arise from preexisting cells

4) hereditary information passes from cell to cell

5) all cells have the same basic chemical composition

6) energy flow occurs within cells

Staining Methods

Staining methods use dyes or labeled molecules to make specific structures easier to see and distinguish under a microscope.

This is done because cells are mostly water and thus transparent

Antibody Structure

An antibody (immunoglobulin) has a Y-shaped structure made of four protein chains: two identical heavy chains and two identical light chains, held together by disulfide bonds.

An antibody's core function is to recognize and bind a specific antigen with high specificity, essentially "tagging" it. Once bound, in the body this tagging can trigger destruction of the antigen (e.g., by recruiting immune cells or activating complement) — it's how your immune system flags pathogens for elimination.

In immunofluorescence, we're hijacking just the binding specificity part of that function, not the destruction part. The primary antibody's job is purely to find and latch onto its target antigen with precision, and the secondary antibody's job is to find and latch onto the primary — neither one is triggering any actual immune destruction in this lab context. We're just using antibodies as very precise "molecular flags" to mark a location, then lighting up that flag with a fluorophore so we can see it under a microscope.

Fluorophore/Fluorochrome

A chemical compound that absorbs light at one wavelength and emits it at another (fluoresces), allowing visualization under a fluorescence microscope.

Primary Antibody

A primary antibody is an antibody raised specifically against the antigen of interest (e.g., a protein on the organelle you want to visualize), and it's the antibody that directly binds to that target.

It usually isn't tagged with a fluorophore itself — instead, its Fc region gets recognized by a fluorophore-tagged secondary antibody, which is what actually produces the visible signal.

Secondary Antibody

A secondary antibody is raised against the primary antibody's host species (essentially treating the primary's Fc region as its own antigen), and it's the one that carries the fluorophore.

It binds to the primary antibody rather than the original target, which is what actually generates the visible fluorescent signal you see under the microscope.

Primary and Secondary Antibodies Cannot…?

Cannot come from the same host species (or the same non-cross-reactive isotype).

If both are raised in the same animal, the secondary antibody has no way to recognize the primary as foreign — it wasn't raised against that species' immunoglobulin, so it won't bind. No binding means no fluorophore gets recruited to the target, and you get no signal, even if the primary successfully found your antigen.

Basically the primary antibody may be able to connect though if the primary and secondary antibody is the same then the secondary antibody will never be able to bind to the primary to actually mark the cellular organelle of interest as antibodies from the same species do not see one another as “threats” to be marked for destruction.

Antigen

An antigen is any molecule (usually a protein) that the immune system recognizes as foreign and responds to by making antibodies against it.

In immunofluorescence, the antigen is simply the specific protein on your organelle of interest — you inject that protein into an animal (like a rabbit), and its immune system produces antibodies against it, which become your primary antibody. Antibodies bind to a small specific region of the antigen called an epitope, not the whole molecule.

Direct Immunofluorescence

uses just one antibody — a primary antibody that's directly conjugated with a fluorophore, so it both binds the antigen and produces the signal in a single step. It's simpler and faster since there's no secondary antibody step, but the signal tends to be weaker because there's no amplification.

Indirect Immunofluorescence

uses the two-step system we've been discussing: an unlabeled primary antibody binds the antigen, then a fluorophore-tagged secondary antibody binds the primary. This is more common because multiple secondaries can attach to each primary, amplifying the signal — making it more sensitive, especially for low-abundance targets. It also lets you reuse the same fluorescent secondary with many different primaries, which is more cost-effective and flexible for labs running lots of different experiments.

Can two of the same species primary antibodies be used?

Generally no — not with standard indirect detection, because secondary antibodies recognize the host species, not the specific primary antibody.

So there would be no differentiation between colors of the nucleus or mitochondria if the same species primary antibody was used for both. The secondary antibody would just recognize the primary antibody as the same regardless of the organelle it was hooked to.

Can two of the same species secondary antibodies be used?

Yes — this is actually completely standard and not a problem, as long as the secondaries target different primary species.

For example a mouse secondary antibody can be made to target the rabbit primary antibody and another mouse secondary antibody can be made to target the goat primary antibody. Thus two different organelles could be stained with two different colors as the secondary antibodies hold the fluorochromes.