Unit 1 Bio 1020 - Microscopy, Cell Organization, & Introduction to Membranes

What are some different types of cells?

Animal cells, plant cells, bacteria, archaea

How can cells differ?

Size, shape, chemical requirements (ex. plants fix CO₂ into sugars, release O₂; Animals can not do this,, take in O₂, release CO₂)

What is a (living) cell? (Cell Theory)

minimal self-reproducing unit of life, transmits a copy of its DNA to its progeny cells when it divides, made of “self-replicating” collection of molecular catalysts (mainly proteins but also RNA), enclosed by a membrane, and driven by free energy released in a complicated network of chemical reactions

In the concentration and individuation of a cell, key molecules are contained in a membrane that defines a space where…

they can interact and function, taking precursors from the surroundings and disposing of waste

Organelles are _____ ______ partitioned off from the rest of the cell’s interior by membranes. They contained specialized set of ______ required to carry out specific functions.

intracellular compartments; macromolecules

All cells (can/cannot) be categorized as a prokaryote or a eukaryote

can

What is multicellularity?

a biological condition in which an organism consists of more than one cell and more than one cell type, allowing the cells to coordinate, specialize, and work together as a single unit

What are some benefits of multicellularity?

Metabolic efficiency, escaper predators, division of labor

How can two cells with the same DNA look and act completely differently?

What is the specialization of function?

Different cells express different genes in the process of differentiation

What allows us to find cells and even visualize cell structures and macromolecules?

microscopes

Who coined the term “cells”?

Robert Hooke in the 1600s

Who first made simple microscopes through which he observed microorganisms

Antonie van Leeuwenhoek (1632-1723)

What are some limitations of cells before microscopy?

1: Cells and organelles are too small for the naked eye; 2: Cells are transparent; 3: Cells lose structure or decompose when isolated; 4: Cell biology is dynamic and active

Microscopy requires _____ ____ ______

magnification and resolution

Resolution is limited by

wavelength of light

Magnification can make visually larger by

changing the angle of light rays coming from an object

Resolution can focus more by

changing the wavelength of light (shorter gives higher resolution) and gathering more light (numerical aperture)

What are the two types of microscopy covered in 1020?

Light microscopy and electron microscopy

What is light microscopy?

Conventional and fluorescence microscopy

What are the two types of electron microscopy?

Transmission electron microscopy and scanning electron microscopy

What does electron microscopy allow visualization of?

Molecules, small cell structures; smaller than 200 n,

What does light microscopy allow visualization of?

Cells/tissues; larger than 200 nm

What are the components of a microscope?

Light source, condenser lens, objective lens, eyepiece lens?

Series of ____________ allows magnification= enlarging

conves lenses

The __________ collects light from the specimen (4x - 100x)

objective lens

Cell Theory simplified

1. All organisms are composed of one or more cells

2. The cell is the basic unit of structure and organization in organisms

3. All cells come from preexisting cells

The eyepiece _________ forms an enlarged image (usually 10x or 15x)

ocular lens

Total magnification = magnification of ________ ___ magnification of ____________

objective lens x ocular lens

Refraction is how the lens bends the light by changing…

speed and angle of the light

The light microscope uses a _______ to provide resolution by _________________

condenser; optimizing the light path

A ______ focuses and shapes the light reaching the specimen, optimizing light path by ______________

condenser; letting in more light and angles

Electron microscopy has (higher/lower) magnification and (better/worse) resolution than light microscopy

higher; better

Light microscopy can reveal larger __________

subcellular components/organelles

______ of convex lenses illuminate specimen and magnify = enlarge image. Resolution is limited by the _____ of light.

Series; wavelength

Electron beams are ________ wavelength

short

What are the two types of electron microscopy?

Transmission EM and Scanning EM

Transmission EM requires a

very thin prepared sample for electrons to pass through

What happens in scanning electron microscopy?

• electrons bounce off surface of sample

• 3D surface

• Specimen thick

• Coated with heavy metal

• Electrons at surface scatter

• Construct details of surface from high intensity signal

What happens in transmission electron microscopy?

• Specimen is very thin

• Stained with heavy metals that interact with biomolecules

• Electrons either pass through (NO stain) or scatter (bounce off) when hit heavy atoms

• Collected by detector

How does microscopy deal with the challenge that cells are transparent?

Bind biomolecules with heavy metals to create contrast for visibility

How does microscopy deal with the challenge that cells are colorless?

Using staining to look at overall morphology of tissue

What are the two types of light microscopy?

Phase contrast and DIC

Refraction

speed of light as it goes through material

In phase contrast and DIC, refraction can be detected and…

differences in speed of light converted to differences in brightness

What is brightfield light microscopy

Bright background with contrasting specimen; ideally suited for specimens with high contrast such as stained samples

What is the halo-effect?

Produced by phase contrast by a sharp changes in refractive index (little light around the cell)

How does fixation work?

Using chemicals or solvents that preserve the structure and morphology of cells, c

What are the two types of fixation?

PFA or MeOH

How do you account for the challenge of cell biology being dynamic and active?

Video record cells in real time, speed up videos and watch what happens

What kind of microscopy should you use to visualize one or a few proteins?

fluorescence microscopy

fluorophores can ________ and _______ light

absorb (one wavelength); emit (longer wavelength, lower energy)

In fluorescence, cells are labeled with ______

fluorophores

What are the two types of molecules used for fluorophores?

Synthetic small-molecule fluorophores and naturally-occuring green fluorescent protein (GFP)

What are synthetic small-molecule fluorophores?

• Small molecules like fluorescein and rhodamine are easily conjugated to antibodies against the protein of interest

• Used as dyes to label cellular structures

What is naturally-occuring green fluorescent protein (GFP)?

a protein that fluoresces used for live imaging

What does GFP imaging require?

Recombinant DNA technique to build transgene to express the protein of interest in cells

Chromophone

The light emitting and light absorbing part of the structure

Fluorophore-conjugated antibodies

Fixed immunofluorescence; uses antibody against histone protein; fluorophore on top of secondary antibody, secondary binds to primary, primary binds to antigen of choice

What is the antigen?

The specific protein in cells

What is the epitope?

region on the antigen that the antibody will recognize

In the antibody, the heavy chain is the _____ domain

constant; specific to species

In the antibody, the light chain is the _____ domain

variable; specific to antigen

How are antibodies built?

From larger heavy polypeptide chains and smaller light polypeptides

Secondary antibodies _____ the fluorescence signal

amplify

Why is indirect immunofluorescence beneficial?

Multiple secondaries can bind to a primary, amplifying the signal

What part of the antibody is introduced into another host to create more antibodies?

fragments of the constant (heavy chain) part

What is DAPI for?

staining the nucleus

In most applications of immunofluorescence, when cells are fixed they are also

dead

In most applications of fluorophore-tagged proteins expressed via a transgene, cells are

living

Transgene

foreign DNA is introduced to the animal or cells in culture, using recombinant DNA technology

Fusion gene produces a fusion protein, which is

our protein of interest that has recombinant DNA to emit fluorescence

How do you find when and where a promoter of interest is active?

By using a transcriptional reporter and putting a GFP after it to see level of expression

What are the advantages of using GFP to find when and where a promoter of interest is?

• GFP is active and nontoxic

• By following protein made, other things could be happening, but by using GFP, you can see how much the promoter is transcribing the gene and where it is

In what scenario would you want to use a fluorescent protein fusion rather than a transcriptional reporter?

When you want to see activity within a cell (i.e. when and where a protein goes)

In what scenario would you want to use a transcriptional reporter rather than a transcriptional reporter?

When you want to see activity of a promoter’s expression is across cells to view which subset of cells have this promoter’s activity

Cytosol vs cytoplasm

cytosol is the fluid, cytoplasm is the entire content within the plasma membrane

Lumen

space encapsulated by internal organelles

Cells can have a lot of _____, but only have one ____, one ______, and one________ (when not dividing)

mitochondria; nucleus; Golgi apparatus; endoplasmic reticulum

Proteins with zip codes called _______________ are recognized by transport factors that bind to the central pore (req energy)

nuclear localization signals

______________ is a selective pore that sits within the nuclear envelope

Nuclear pore complex

Two ______ membranes form the nuclear envelope.

concentric

What types of proteins need to be transported back into the nucleus after translation by ribosomes in the cytoplasm

transcription factors, DNA polymerase, anything that has to do with DNA replication

Rough ER is studded with ribosomes that are actively translating proteins while…. (These proteins are then modified and shipped to the Golgi)

co-translationally inserting them into the ER lumen

Lysosomes have a specific, acidic pH, which allows them to

contain a variety of digestive enzymes

The acidic lumen of the lysosome _______ the digestive enzymes

activates

Peroxisomes contain a variety of enzymes to carry out _________ reactions related to lipid __________ and ________, as well as the degradation of harmful __________

oxidative; biosynthesis; degradation; hydrogen peroxide

Only ______ and ________ come from bacterial origin

chloroplast and mitochondria

___________ are derived from the ER and can be isolated via centrifugation

microsomes (vesicles)

SDS page is an ionic detergent that

binds and denatures proteins; also imparts a negative charge to all proteins in sample

___________ is added to break disulfide bonds

Mercaptoethanol

Secondary antibodies typically covalently linked with ___

HRP (horseradish peroxidase) marker that illuminates when substrate is added

What can pellet at low speed centrifugation?

whole cells, nuclei, cytoskeleton

What can pellet a medium speed centrifugation?

mitochondria, lysosomes, peroxisomes

What can pellet at a high speed centrifugation?

closed fragments of endoplasmic reticulum, other small vesicles

What can pellet at a very high speed centrifugation?

ribosomes, viruses, large macromolecules

Integral membrane (transmembrane proteins

penetrate the lipid bilayer; have domains that protrude both sides of the membrane

Peripheral membrane proteins

located entirely outside of the lipid bilayer, yet associated by noncovalent bonds

Lipid-anchored membrane proteins

located outside the lipid bilayer but are covalently-linked to a lipid molecule in the bilayer

The extracellular surface of many cells have a thick

carbon sugar coating