Cellular Biochemsitry Year 1

What is a life?

Ability to self replicate

Why is a cell a unit of life?

Contains all the genetic information and metabolism to self replicate

Describe the flow of energy in a cell

Metabolism: cells must harness free energy and use building blocks.

Describe the flow of genetic information in a cell

Genetic information must be replicated in order to replicate a cell, and each cell has the entire set of genetic information, meaning it can generate a whole organism

What type of system is a cell and why?

A closed system, which is bound by a lipid membrane

What is a multicellular organism in terms of cells?

A colony of cells at the service of the germ line

What is the shape and size of a multicellular organism determined by.

Cell growth, division, movement, attachment and contraction

Where did all cells originate from?

Life appeared only once, so all living organisms come from a single common ancestor

How did Darwin study relationships between individuals?

Looked at pentadactyl limb structure, noticing similarities between species, this is comparative anatomy

How can we study similarities between cells?

Look at similarities in the DNA sequence.

Describe prokaryotes

Made up of bacteria and archaea, simple internal organisation and they are a single compartment surrounded by a plasma membrane.

Contain a single circular DNA molecule (1 chromosome)

Some contain an additional plasm of

Generally unicellular

No visible nucleus, DNA is folded into a nucleoid

Describe the structure of eubacteria

• All have inner plasma membrane, some have an outer one

• Surrounded by a peptidoglycan cell wall

• Gran +ve gave a thick peptidoglycan cell wall and gram -ve have a thin peptidoglycan cell wall and lipopolysachharide layer.

What is peptidoglycan made up of and why?

Proteins and oligosaccharides, sugars are used in cell walls as they can form hydrogen bonds which are very strong.

Describe the structure of archaea

• No nucleus

• Resemble eukaryotes as have similar genetic information handling machinery.

• Replicate eubacteria in shape, size and organisation.

• Lack peptidoglycan cell walls, their are made of polysaccharides and proteins

• Cell membranes contain ether lipids, not ester lipids

What are the types of archaea and where do they live?

• methanogens - anaerobic conditions, produce methane from respiration, usually in ruminant stomachs

• halophiles - live in high salt environments such as salt lakes

• thermoacidophiles - live in high heat and acidic conditions

• psychrophile - live in cold environments

What are the two extremophile survival hypotheses?

• Early Earth Hypothesis

• Competition Hypothesis

Describe the Early Earth Hypothesis

Extremophiles thrive in conditions similar to early earth, they could have been the first life form to evolve, they may have evolved in conditions too harsh for other life forms to occupy

Describe competition hypothesis

• Extremophiles evolved in all environments but were outcompeted by eubacteria and eukaryotes

• They now only populate habitats with no competitor

What ar lokiacheota?

Closest relatives to eukaryotes, they make protrusions, which hints to complex evolution

Describe eukaryote structure

• larger cells with a complex internal structure

• Membrane bound nucleus and extensive internal membranes that house other organelles

• Plasma membrane

• Contain cytosol, a liquid between organelles

What are the common features between eukaryotes and prokaryotes?

All use DNA as their genetic material

What is a common features between eukaryotes and eubacteria?

They both have ester lipids in their plasma membrane

What is a common features between eukaryotes between eukaryotes and archaea?

They share the same machinery to handle genetic information

What functions do cells do?

Replicate

Maintain a constant energy flux

Establish larger multicellular organisms

What are anabolic reactions?

Build complex molecules form building blocks

What are catabolic reactions?

Break down complex molecules

Why are catalysis reactions better than combustion reactions when breaking down compounds (catabolic reactions)?

Catalysis allows energy to be released in controlled stages, rather than all at once as it would be lost as heat

It produces intermediates that can be used

Energy released can be used to fuel another reaction that is not normally energetically favourable, this is reaction coupling.

What is an organotroph?

Feed on organic substances produced by others

What is a photolithotroph?

Use Co2 as a carbon source and sunlight as an energy source

What is a chemolithotroph.

Used reduced inorganic compounds as an energy source

How can eukaryotes move and change shape?

They contain an internal cytoskeleton (made of filaments) that exert a force on a specific part of a cell

Describe apoptosis

• Programmed cell death, which occurs when a cell receives a death signal, due to something wrong in the cell

• Death signal causes the cell to produce proteins required for self destruction.

How do animal cells produce their own environment?

They secrete collagen

How so cells communicate?

• They exchange small molecules through gap junctions

• Detect signals via receptors

• Evoke responses via signalling pathways

• Signals can also come from an external environment

What is the function of microfilaments?

Cell shape and movement

WHat is the function of microtubules?

Organelle and chromosome movement

What is the function of intermediate filaments?

Mechanical strength

What are microfilaments made of?

Actin monomers

What are microtubules made of?

Alpha and beta tubulin dimers, which make up 13 protofilaments

What are the thermodynamics of filament assembly?

It is always favourable to add more units

What limits filament growth?

The monomer concentration. Polymer is assembled until the critical concentration is reached.

Describe the assembly of actin filaments

Assembles from monomers of Gactin. This requires nucleation. There is self assembly, g actin becomes ATP bound and polymerisation occurs, this is elongation. Accessory protiens form caps, branches and bundles

Describe treadmilling in F-actin

At the pointed end, dissassebly occurs and g actin breaks off which releases ADP bound actin

At the barbed end, ATP bound actin binds.

How does actin filaments control cell movement?

Membrane protrusions are formed as actin attaches to underlying surface

What are the actin regulators?

Capping proteins block assembly and dissasembly

CapZ blocks the +ve end

Tropomodulin blocks the -ve end

Nucleator proteijns help nucleation by holding g-actin subunits together

Polymerising proteins speed up the addition of subunits

Branching proteins drive meshwork formation

What is chemotaxis?

Actin regulators change the shape by cell signalling and sensing chemical mechanical queues

How can actin change the shape of a cell?

Actin can branch or extend the filament, which means protrusions can be produced

How can atomic force microscopy be used to look at actin?

Can determine what happens when forces are apploied. When no force applied, the actin filament is extended. When force is applied, the space becomes constrained and actin molecule branches, to increase thickness, allowing the molecule to become increasingly dense

What is a molecular motor?

Motor proteins that can work to transport organelles and vesicles, as well as helping with muscle contraction

What are myosins?

ATP driven motors that move along actin, they ‘walk’ towards the negative end

How are actin and myosin used in cytokinesis?

They make up a contractile ring that helps divide the cell

What are the two actin like proteins in bacteria?

ParM forms a spindle like structure that separates plasmids during DNA replication

MreB helps with cell shape.

What are the likely origins of actin like proteins in bacteria?

Probably a result of horizontal gene transfer (bacterial transformation) where a gene was encorporated into the genome

Describe the structure of microtubules

Made up of alpha and beta tubulin molecules that form dimers. These dimers hydrolyse GTP

These dimers assemble into protofilaments, and 13 protofilaments make up a microtubule

What sort of stability/instability do microtubules undergo?

continous dynamic instability. Polymerisation and depolymerisation is not constant like in microfilaments. There is a period of catastrophe, when there is a lot of depolymerisation and shortening of the microtubule. This is followed by rescue, where there is a constant repolymerisation. Polymerisation and depolymerisation occir at both + and - end.

What does a microtubule do in the microtubule organising centre?

Responsible for organising the mitotic spindle

Which ends of microtubules are in the MTOC

negative ends

Which way does kinesin walk along a microtubule?

From -ve to +ve

Which way does dyenein walk along a microtubule?

From +ve to -ve

What do motor proteins do along microtubules?

transport vesicles and organelles

Describe the structure of flagella in eukaryotes

Base is made up of 9 triplet microtubules, and the top is made up of 9 doublets

How is the beating movement done in the eukaryotic flagella?

Using axonemal dyenin, which is an ATP dependent motor, this causes the wafting motion

Describe bacterial flagella and the mechanism of motion

Made of flagellin which is made up of 11 protofilaments that give it a rigid corkscrew shape due to a costant bend. Flagellar motor at the base of the flagellum is a rotating motor that provides propulsion force

What proteins are used in bacterual cytokinesis, what are they like?

FTS-Z, they are tubulin like

Describe intermediate filaments

Mechanical strength, do not have any intrinsic polarity, no nucleotide binding so no treadmilling, no motors, very stable so need a lot of energy to dissassemble, unique to metazoans

What are the main intermediate filaments?

Nuclear lamins, keratin, vimentin

Describe the structure of intermediate filaments

Conserved alpha helical rod domains that form a coiled-coil structure due to hydrophylling and hydrophopic regions on amino acids

How to intermediate fiaments interact?

They form dimers that form tetramers (which are symmetric and not polar), these assemble into protofilaments, which assemble into protofibrils and then fibres

What types of movements depend on the cytoskeleton?

Intracellular movement (changing shape)

Cell movement

Chemotaxis (movement in response to external factors)

Movement outside cells

Flagellar movement

Describe how to prepare a cell line

Culture cells in a nutrient rich media

Can use primary cultures to study cell differentiation

Transformed cells can grown indefinitely

Cells produced can be differentiatied, multipotens or totipotent

What are the benefits of a cell line?

Cultured cells offer purity and abundance

One cell type can be grown

Can grow large quantities

Cells can adhere to transparent surfaces, which is useful when making slides.

Cells can have foreign DNA transferred, such as GFP which helps with visualising proteins

Can edit DNA using CRISPR tech

What is forward genetics?

Finds what genes are responsible for phenotypes, by looking at mutations

What is reverse genetics?

Removes genes to see what it does, looks at phenotype changes if genes are removed.

Example: In mice, NESP4 removed and mice became death with age, because vibrations kill inner ear cells unless the nucleus is fastened to the cytoskeleton

What is genomics?

Screens every gene for a phenotype. Can use knock out technology and CRISPR collections

What is in vitro constitution?

Used to purify tubulin from pig brains as they contain lots of microtubules

Describe how tubulin is purified

Depolymerised using cold and no GTP, centrifuge and keep soluble fraction.

Then repolymerise with warm and GTP, centrifuge and keep pellet.

Repeat this procrss to reconstitute the reaction in vitro

How can antibodies be used for analysis?

Antibodies bind to specific immunoglobins, and can be stained to show presence

How can antibodies be produced?

Inject animal with protein and it will make specific antibodies which can be isolated

How can light microscopy be used?

Light passes through samples and shows everything, you can stain specific parts.

Example: DAPI stains DNA

What is cell theory?

All animals and plants are made of individual cells

What is cell compartmentalism?

Process of separating a cells interior into distinct compartments in order to contain specific conditions in concentration

What do transport proteins do in the cell membrane?

Allow the passage of nutrients and cofactors into the cell

What are the two main types of proteins?

Membrane and secreted

What is a signal sequence?

A short peptide that signals where the protein should be taken in the cell

What does a signal sequence do?

Directs the ribosome to the translocon

What is the translocon?

A protein complex that moves proteins between cell compartments

Which two orientations can the translocon insert proteins into the membrane?

Monotopic and polytopic

What are the functions of proteins in the cell membrane?

Act as signal receptors and ion channels, control shape and organisation of membranes by regulating membrane curvature

What is the origin of the ER?

Derived from ancestral cell membrane

What are the origins of mitochondrial and chloroplast cell membranes?

Endosymbiosis, engulfment of bacteria by an ancient archaea

Why do mitochondria and chloroplasts require protein transport across membranes, why is this difficult?

They have minimal genomes, most proteins are in the nuclear encoded so must be imported from the cytoplasm, this is difficult as mito have double membranes and chloroplasts have triple, so more signals are required to allow transport into the organelle

What are organelle targeting systems?

The cytoplasm is the default localisation for proteins without a signal, so organelle proteins require targeting or sorting signals to be encoded into their amino acid sequence. Cells require mechanisms to recognise and interpret sorting or targeting signals

Describe the structure and function of the nuclear envelope

DNA is wrapped in a double membrane that is connected to the ER

There is a protein meshwork (the nuclear lamina) that sits between DNA and the innner nuclear membrane, which gives support

There are many nuclear pore coplexes (NPC’s) that control what enters and exits the nucleus, these substances must have the correct signals

The NPCs are bidirectional gateways

Proteins form a gel like barrier so no cytoplasmic proteins can enter and nuclear proteins cant leave unless they have the correct signal

Describe the regulation of nuclear transport

GTP switches are a regulatory mechanism

Ran is a switch protein which binds to GTP or GDP

Ran binds to GTP in the nucleus

Ran hydrolyses GTP to GDP in the cytoplasm

RanGTP binds to nuclear import receptors which promotes protein release

RanGDP is released from import receptors which allows protein binding

Describe the signal hypothesis discovery

Antibodies normally have light and heavy chains. They have an N terminal signal that is cleaved before they assemble.

When in vitro, the light chains were larger than normal, as the N-terminal protein had not been cleaved from the protein. When added to ER, the signal is removed.

This showed the signal protein targeted the antibody to ER.

Describe signal hypothesis

During synthesis of a protein, a signal peptide is formed which directs the ribosome to a channel in the ER membrane.

The Ribosome, mRNA and growing protein bind to the ER surfsce, synthesis stops until the signal is recognised by translocation machinery

The protein passes through the ER channel and the signal peptide is removed.

The protein is released into the ER and secreted to the correct area.

Describe single sequence-dependent membrane protein insertion into ER

Signals stop proteins forming a blob before ribosome binds to the mebrane, SRP prevents synthesis until the protein has reached ER and can be properly encorporated into the cell membrane

Why is the ER a useful folding compartment?

Contains enxymes that allow disulphode bond formation, which is crucial for some proteins. This also releases toxic products so compartmentalism stops it getting into other cell components

ER can make complex sugards to add to proteins at an asparagine residue (glycolisation)

How does the golgi apparatus transport proteins?

Portions of the golgi membrane break off forming vesicles at the trans face

What is the endocytic pathway

Lysosomes contain acid hydrolyses for hydrolysis of macromolecules.

Macromolecules must be moved to the lysosome, so there needs to be signals in the ER, there are specific receptors that trigger movement to lysosomes in vesicles