lectures 22-33 Flashcards
centrosome
centrioles and microtubules
interphase G2
2 centrosomes visible (but higher plants and most fungi lack centrioles)
nucleus intact and chromosomes not visible by light microscopy (only visible by FISH)
active
relaxed chromatin: transcription factors can access genes so protein production
FISH (fluorescent in situ hydridisation)
cells fixed and permeabilised with detergent to form monolayer
incubated with fluorescent oligonucleotide primers specific for individual chromosomes
primers hybridise with targets, bind to target by base pairing
chromosomes become painted
prophase
early: centrosomes move to poles
chromosomes condense to visible threads
nuclear membrane disintegrates into small vesicles
nucleus surrounded by microtubules
late: each chromosome composed of 2 sister chromatids held together at centromeres
microtubular spindle fibres grow near centrosomes, some extend across poles, others attach to chromatids at kinetochores
metaphase
chromosomes align in middle between poles
sister chromatids remain attached by centromeres
anaphase
sister chromatids separate into separate independent chromosomes, centromere splits
each centromere attached to spindle fibre and moves to poles
cell elongates and spindle elongates
cytokinesis starts
telophase
chromosomes uncoil and become less distinct
nuclear membranes form around daughter nuclei
nucleoli reform
spindle fibres depolymerise: becomes less distinct and disappears
cytokineses complete: separates 2 daughter cells
interphase G1
after cytokinesis
chromatids in daughter cell double up to give chromosomes in S phase
amount of DNA different to G2
cell cycle
M
G1 - 1st gap, repair damage, growth, duplication
S (synthesis) - chromosomes doubled also centrioles and other organelles doubled
G2 - 2nd gap, ready for mitosis, proteins that condense chromosomes
controller goes round clockwise
experiment for identifying proteins involved in cell division
stimulate egg into growth but not division
fertilised = growth and division
compare proteins between resting, growth, growth and division
spot proteins involved in division (cell cycle controllers)
add radiolabelled methionine at time 0, sample between 25-127 min, place on gel
apply current so separate in size
boil eggs (in presence of SDS negatively charged detergent to keep protein soluble, and reducing agent)
unfertilised (resting): protein X,Y,Z drug stimulation (growth): A,B,C proteins fertilised (growth and division): A accumulates then levels fall and rise again and fall, so named cyclin because cycles with cell cycle, characteristic of dividing
Cyclin A….
expression rises and falls in expression levels after fertilisation (dividing cells)
destroyed every time cell divides
peaks just before cell division
controller, high concentrations stimulate mitosis
where was the cyclin controller first identified?
fertilised sea urchin eggs
cyclin levels of complexity
regulatory, makes decisions of what to phosphorylate and then kinase (CDK) phosphorylates
inactive unless have partner (CDK)
additional layers include cyclin/CDK inhibitors and activators, plus their regulation
cyclin/CDK complexes controlled by destructive phases that reduce their conc after performed function (regularly destroyed)
kinase
phosphorylates
transfer phosphate group
CDK
cyclin dependent kinase
cyclin + CDK process
CDK + cyclin in inactive heterodimeric complex that is prepared to be activated
modifying enzyme makes complex prepared for activity
then complex either activated or inhibitedby CDK inhibitor/activator
if activated, targets chosen by cyclin are phosphorylated by CDK
then complex is destroyed by proteolysis in proteasome (cytosolic proteolytic complex)
G1 cyclin-CDK complexes
3 different CDKs and 2 cyclins, so multiple complexes
prepare cell for S phase
target range of proteins that allow progress through G1 and prepare for S
stimulate and promote expression of S phase cyclin complexes
S phase complexes
only 1 cyclin and CDK as 1 complex
phosphorylates targets which control chromosome replication
conc of S phase controller rises and progress through S phase, then destruction and replaced by G2 complexes
G2 complexes
prepare for mitosis and modify and active spindle fibre formation
controllers in the cell cycle?
G1
S
G2/M
cyclins+CDK
simplest organism has….
2 cyclins
experiment to find out evolution of cyclin+CDK
add flexible linker (Gly4, Ser) between 1 cyclin and 1 CDK
conc. of cyclin and CDK is artificially high so drives them to heterodimerise (join)
Cdc13 (CDK) used because does most work in Sz.pombe
the artificial complex was expressed in Sz.pombe and all others were deleted
cells grew slightly slower but still worked
low conc of the complex phosphorylated G1 targets allowing entry to S phase (high affinity targets, for CDK)
high conc of complex phosphorylates G2/M targets stimulating mitosis (low affinity targets)
so can run with 1 CDK and 1 cylcin
therefore: evolved to form new combinations that performed at diff stages of cell cycle resulting in complexity we see in modern organisms
stationary phase regulation of cell cycle
leave cell cycle and enter quiescent phase
cells stop dividing but if not left too long, can re enter cell cycle if diluted into fresh growth medium
regulated to either leave or enter cell cycle
unicellular cells……. than our cells because…
response better to an env. stimulus
can move away from a bad env but we can’t so our cell cycles are highly regulated
G0 and cancer
cancer cells can enter G0 phase and return to cell cycle, so if kill all rapidly growing cancer cells, may be some cells left in G0 that can’t be targeted and tumour may reform later on - relapse
Radiotherapy targets rapidly growing cells and chemotherapy targets in S phase
cancer cells go quickly through G1 so can’t repair in G1
G0 differentiation and proliferation
cells can differentiate in G0
some differentiated cells remain post-mitotic and don’t re-enter cell cycle
other differentiated can be stimulated by mitogenic signals to re-enter cell cycle and replicate (fibroblasts, lymphocytes)
restriction point: cell is committed to cell cycle and can’t go back (but can return to G0 before this point)
mitogenic signals
EGF ligand (epidermal growth factor) binds to EGFR (EGF transmembrane receptor) dimerises the receptor - 2 kinase domains phosphorylate each other - P is negative so alters the size and charge and contrains flexibility activation
activated EGFR bound by adaptor molecule that recruits and activates cytosolic membrane-bound Ras enzyme (bound to membrane by lipid anchor)
signal transduced from extracellular to intracellular
Ras recruits Raf (kinase so phosphorylates) to membrane and signal passed via intermediates to MAPK (so activated)
signal transduced across cytosol
activated MAPK translocates to nucleus, stimulates expression of early response genes c-FOS and c-JUN
so MAPK acts as transcription factor
c-Fos and c-Jun are transcription factors that induce expression of delayed response genes including cyclins and their partner CDKs
so triggers re-entry to G1 in cell cycle
mitogenesis - induction of mitosis
mitogenic signals: main points
activation of growth factor receptors recruitment of Ras signal transduction (Raf to MAPK) induction of early response genes induction of delayed response genes expression of G1 cyclins and CDKs that return a G0 cell to G1
MAPK
mapkinase
shutting the mitogenic signal off
inappropriate growth signals cause unchecked proliferation (have to shut down or=cancer)
signal cut off by lysosomal targeting and destruction of activated growth factor receptors (EGFR)
Ras cannot be recruited and so on, so shuts down expression of genes
mutational activation of receptors
25% of breast cancers have a mutation in the transmembrane domain of c-erbB1 growth factor receptor
self-activating, dimerising and auto-phosphorylating in absence of growth factor
so causes unregulated proliferation
so has ligand independent manner and forced to divide
mutational activation of Ras
15-30% of all cancers have mutated Ras that permanently activated
so MAPK stimulated in absence of growth factor causing unregulated proliferation
so no need for ligand
mutational activation of Raf
66% of malignant melanomas have mutated BRAF gene that produces permanently activated Raf
so MAPK activated
viral subversion
viral oncogenes products v-JUN and v-FOS mimic action of c-Jun and c-Fos
what might cause genome instability?
deregulation of the cell cycle, running at full speed so no time to proof-read newly replicated DNA before daughter cells are separated
failure of check point means mutations carried to next generation
cell cycle brakes operate in……..
and…..
G1 - repair
G2 - some recognise misincorporation of nucleotides
repair is operated by…
2 proteins
Rb and p53
which slow down the cell cycle so allow repair
childhood retinoblastoma treatment
laser surgery/cryotherapy - small tumours
radiotherapy - local or larger tumours
chemotherapy - tumour spread beyond the eye
surgical removal - if above fail
childhood retinoblastoma hereditary
sporadic
normal Rb+ allele from 1 parent and defective from other
somatic mutation inactivates normal allele so Rb- cell
2 normal Rb+ alleles
2 separate mutations required to inactivate each
rare
Rb
tumour-suppressor gene
stops cells proliferating uncontrollably
inhibits formation of retinoblastoma
Rb protein regulates restriction point (only exists because of Rb)
inhibits G1 controllers so extends time of G1 so more time to check damage (keeps cyclin+CDK complex inactive)
there is a fixed amount of Rb in cells but G1 controllers always manufactured so there’s excess of controllers at restriction point, enough to push into S phase (cyclin+CDK override Rb control)
p53
tumour suppressor
stops genome instability
TP53 mutations associated with 50% of cancer
normally inactive because degraded by proteosome so conc. normaly low
DNA damage makes it stable so activates it (acts as transcription factor)
activates gene expression of proteins that inhibit cyclin/CDK complexes
stops cycle (arrest) or apoptosis
mostly in G1, sometimes G2
works upstream of G0
viral subversion of Rb and p53 human papillomaviruses
E6 protein inhibits p53 so not able to apoptosis
E7 protein inhibits Rb because take restriction point away so no time to check damage
apoptosis process
signal received
mild convolution, chromatin compaction, cytoplasmic condensation
nuclear fragmentation, cell blebbing, cell fragmentation
phagocytosis
no inflammation because no release of cytoplasm
necrosis
die through tissue damage
dying cells swell and burst so inflammation
intracellular constituents released into extracellular matrix
what model organism was used to understand apoptosis?
C. elegans
classes of protein function in C.elegans apoptotic pathways
cells that die by apoptosis would have become neurones mostly
ced-3 mutation : all cells survive, no apoptosis
so some proteins required for cell death, wild type promotes apoptosis
ced-9 mutation: all cells die, so protein suppresses apoptosis
so some proteins required for cell survival
Caspases
effectors for apoptosis
cleaves proteins of the nuclear lamina and cytoskeleton, leading to cell death
cleaves targets at site just C-terminal to aspartic acid residues
normally kept inactive by trophic signals from neighbouring cells
process of suppressing apoptosis
trophic signal from neighbouring cells binds to receptor
keeps procaspase inactive
process of activating apoptosis
no trophic signal
so active caspase
substrate cleavage and cell death
triggers for apoptosis
external: lack of trophic signals (stop telling cells to live)
recognition of stress, virally-infected cells
internal: recognition of irrepairable DNA damage
developmental: remove webbing between fingers, foetal development, remove neurones, highly regulated
the ……… is so high in a cell that………….
even the cytosol is…..
conc. of proteins
it’s close to the limit of solubility
packed and so gel like and not a liquid
cytosol
aqueous component of the cytoplasm (fluid phase)p
site of protein synthesis and metabolic pathways
peroxisome
sites for oxidative reactions
vacuoles
turgor or protein storage/degradation
we can visualise subcellular organelles…….
in vivo using dyes or Fluroscent Proteins such as GFP
how do proteins know where to go?
sorting signals that are part of the protein
can be:
short peptides at N- or C- termini (removed after use or kept for use again)
3-dimensional domains
other molecules attached to protein that not part of sequence itself so post-translational modifications (sugars/lipids)
what happens to sorting signals?
recognised by specific receptors which trigger transfer of protein to correct destination
every organelle uses different receptors and sorting processes because different signals
modes of protein transport
gated transport: physical barrier/gate e.g. nucleus
transmembrane transport: need channels to cross mitochondrial/ER membrane
vesicular transport: surrounded, packaged, fuse
gated transport into the nucleus
large aqueous nuclear pore complexes (NPC)
storage of chromatin, large volue in and out nucleus
transcription factors in and out
pores very abundant
structure of a nuclear pore complex
proteins line ring
rod-shaped proteins - linear and flexible
cytoplasmic ring with cytoplasmic filaments
nuclear basket in nucleoplasm
central transporter in between proximal filaments
massive
made up of many copies of different nucleoporins (proteins)
FG-nucleoporins line the channel, nuclear basket and cytosolic fibrils (F and G AA residues, don’t acquire 2ndary structure but stay in filament)
what sized molecules can rapidly diffuse between cytoplasm and nucleoplasm?
which diffuse slowly?
which can’t enter?
small molecules 5kDa or less
proteins 20-40,000 Da
proteins >40 kDa, RNA, ribosomes
diffusion barrier
unstructured regions of NPC proteins forming tangled network and blocking passive diffusion of large molecules
filaments oscillate and collide so repel materials trying to get in
NLS
nuclear localisation signals
rich in lysine and proline in any position on protein as long as exposed to surface
importins
NLS receptors
cytosolic nuclear import receptors
each responsible for set of cargo molecules
nuclear import
importin binds NLS on cargo protein and binds FG repeats in FG-nucleoporins of fibrils and channel filaments in nuclear pore
transient interations with FG anchor points
repeated binding and dissociation so climb along pore
importin receptors then disengage from cargo in nucleus
NLS not cleaved off
Ran-GDP
Ran-GTP
in cytosol
in nucleus
importin letting go of cargo
Ran binds to importin - triggers conformational change
release protein that was in importin
importin still bound to Ran GTP
importin to cytoplasm, GTP hydrolysed to GDP
so lets go of importin
why is there asymmetric distribution of Ran-GTP and GDP?
because of proteins that can switch Ran on or off
Ran-specific GEF - in nucleus, guanine nucleotide exchange factor (exchanges GDP with GTP, so makes GTP), tightly bound to chromatin in nucleoplasm
Ran-specific GAP - in cytosol, GTPase activating protein (promotes hydrolysis of GTP to GDP), bound to importin
ER functin
lipid synthesis e.g. in adipose tissue
protein translocation - start journey in ER, through translocation pore, proteins acquire native structure in lumen, proteins N-glycosylated (sugar attached to asparagine residue), proteins degraded if fail to assemble
How do proteins enter the ER and secretory pathway?
secretory proteins carry N-terminal signal sequence that targets them to ER (while protein still being made)
co-translational: occurs during translation, recruits receptors that take ribosome with chain to ER
leads to docking of ribosome-nascent chain complex into ER membrane
signal sequence removed once protein in ER so it’s 1-directional
Golgi function
protein and lipid modification (glycan processing, tyrosine sulfation)
protein packaging and sorting (to outside/plasma membrane/lysosomes)
modifies AA residues, decision on where protein go and sorted to diff secretory vesicles
the lumen on the secretory pathway is….
topologically equivalent to the outside of the cell so connected and organelles interconnected
once in the ER, protein doesn’t need to cross any membranes to be secreted
endosome?
vesicle
late endosome become lysosomes
sorting in biosynthetic secretory pathway
proteins for plasma membrane secretion don’t need signal because this is default
proteins for intracellular destinations (lysosomes) need sorting signal
lysosomes
intracellular endpoint of secretory pathway
degrade particles/organisms/proteins/organelles
rich in hydrolytic enzymes
low pH
targeting hydrolytic enzymes to lysosomes
all lysosomal enzymes are glycoproteins (have sugar-glycan)
mannose residues on glycans are modified to mannose 6-phosphate (M6P) - targeting signal
M6P receptors in trans Golgi network (TGN) membrane
binding triggers process that recruits proteins (specific adaptor proteins (AP) and clathrin) that bend Golgi membrane into bud
dynamin protein wrings the neck and structure gets smaller till pinches off vesicle (GTP hydrolysis)
loses clathrin/AP coat and directed to late endosome
M6P receptor off M6P containing protein (from pH)
receptors back to Golgi (vesicle by retromer not clathrin)
clathrin
force membrane to bend
cage contains spherical membrane
what allows lysosomal enzymes to have M6P signal?
have 2nd (3 dimensional) targeting signal that recognised by enzyme in early Golgi, tells to attach phosphate at position 6
has signal patch, recognised by GlcNAc phosphotransferase in early Golgi
binds sugar (UDP-GlcNAc) which carries phosphate
bind phosphate to position 6 on mannose
2nd enzyme (phosphodiesterase removies GlcNAc leaving phosphate bound to mannose residue
lysosomal storage diseases
Gaucher’s disease - lack of glucocerebrosidase which breaks down glucocerebroside
pleiotropic phenotype
treat by giving enzyme
I (inclusion) cell disease (mucolipidosis II) - multiple lysosomal enzymes missing because no GlcNAc phosphotransferase so undigested material, growth ceases
Tay Sachs disease - lysosomal accumulations of gangliosides in neurones (limp baby)
Hunter and Hurler disease - similar to I disease
extracellular matrix (ECM)
major product of secretory pathway
material that surrounds animal cells
produces variety of structures e.g. bone, teeth, tendons,exoskeleton
can also regulate behaviour of resident cells
dynamic
makes tissue function
influences survival, development, migration, shape, proliferation, function of cells
ECM is abundant in….
spaces around cells are filled with….
what is secreted into the gel?
molecules of the ECM are produced…
connective tissues (CT) loose and dense
hydrated polysaccharide glycosaminoglycans often linked to proteins to form a proteoglycan gel (hydrophilic)
collagen, fibronectin, elastin are secreted into this gel
locally, cells make own matrix
glycosaminoglycan (GAG)
chains are repeated units of negatively charged disaccharides
attract cations so water sucked into matrix (osmotic effect)
form linear chains
occupy large volume relative to their mass so can absorb water and create turgour (swelling)
4 main groups because of different sugars, linkages, sulphate groups
4 main groups of GAGs
hyaluronan
chondroitin and dermatan sulphate
heparan sulphate
keratan sulphate
most GAGs can be linked to proteins to form…
proteoglycans (PGs)
(sugars linked to proteins)
linked by O-glycosylation because attached to OH group
very large complex structure
varied nature of GAGs and PGs means…
pore sizes and charge densities in gel vary which influences turgor and what cells can pass through ECM
PGs can bind…
signalling molecules to enhance/inhibit their activity to affect nearby cell proliferation and modulate inflammatory responses
proteases (degrade matrix to allow cells through) to concentrate them/delay release/inhibit
aggrecan
proteoglycan with 100+ GAG chains that connects with another GAG (hyaluronan)
hundreds of chains on protein on hyaluronan
sucks in lots water so shock absorber
mostly in cartilage
withstand mechanical stress in joints
in collagen (mesh of proteoglycans with proteins through them)
major proteins of ECM
collagen (most abundant)
elastin
fibronectin (links between matrix and cells)
laminin (basal laminae only)
secreted by cells in ECM
collagens/elastin give strenght/resilience and anchored by sticky fibronectin/laminin to integrins of cells
fibroblasts in connective tissue
produces collagen and other fibres
secretion rate very high
collagen
most abundant protein in vertebrates
20 types of collagen
main types in connective tissue: 1,2,3,5,11 (fibrillar collagens)
collagen trimer can cross-link to others to form fibrils
type 9 and 12 are fibril-associated collagens (links fibres together)
type 4 forms mesh in basal laminae
trimeric (3 chains)
stiff triple stranded helical structure with lots H bonds between 3 subunits
hydrophobic polypeptides (alpha chains)
glycine every 3rd residue with proline and hydroxyproline between
inflexible helix
fibrillar collagens
made in cells as precursors (procollagens) so can get out ER of cell to outside
N-and C-terminal propeptides (part of protein that help fold then eventually removed) - not present in mature extracellular collagen
proteins undergo modifications from site of folding to deposition in ECM
if no propeptides, chains would try to pair leading to dimers and trimers that not folded correctly
synthesis of fibrillar collagen
in fibroblasts of loose CT, osteoblasts, chondroblasts or bone/cartilage
C-terminal propeptides on collagen acts as nucleation point that brings 3 chains together and twist
disulfide bonds between chains between C-terminal propeptides
globular N- and C- propeptides ensure triple stranded regions don’t get too close to prematurely form fibrils in ER and Golgi (keep trimers apart), otherwise will become big and can’t secrete
prolines become hydroxylated to facilitate intra-chain hydrogen bonding
lysines also hydroxylated to help cross linking of tropocollagens outside the cell
chains glycosylated (sugar added)
propeptides removed just before secretion so can H bond chains, tropocollagens cross linked to form fibrils
fibril formation in fibripositors close to plasma membrane
fibripositors
where formation of fibrils occurs
tropocollagen
processed procollagen without propeptides
form intra and inter molecular crosslinks through hydroxylysyl residues to generate a fibril
trimers form H bonds with others to form super cable-like structure - stacks of fibrils
in some tissues like tendons, fibrils…
aggregate as parallel bundles to form a fibre
fibrils in ECM of connective tissue in skin are arranged like _____ to…
wickerwork
resist stress in all directions
stacked at 90 degrees
collagen fibres in bone
plywood arrangement that doesn’t allow stretching
withstand pressure
basal laminae
specialised ECM
underlies epithelial tissues and surrounds other cell types
influences cell polarity, metabolism, survival, growth, differentiation, repair (scaffold along which regenerating cells can migrate)
made from type 4 collagen, PG perlecan and proteins laminin and entactin
(combination of collagen and glycosaminoglycans)
collagen disease
defects collagen or post-translational modifications
Elastin in ECM
hydrophobic elastic protein
extensive crosslinks that determine limit of extension
in ECM of arteries
can be stretched
Fibronectin
adhesive dimeric protein of ECM, makes connections
2 monomers held by disulfide bonds, acts as glue
multiple domains for binding to other ECM molecules
cytoskeleton and fibronectin
cytoskeleton contract and pull on fibronectin in matrix to create tension
expose other fibronectin binding sites
control over composition of ECM
ECM degradation
by proteases
need to degrade to let cells through
needed in tissue repair and remodeling
strength in cell structures comes from:
ECM
internal cytoskeleton
cell-cell adhesion
cytoskeleton function
mechanical strength organelle movement (along microtubules) anchor for cell-cell junctions chromosome segregation and cytokinesis cell movement muscle contraction
cytoskeleton structure
scaffolding microtubules actin (shape and movement) intermediate filaments (mechanical strength) associated proteins
microtubules
non covalent polymers of tubulin linear arrays of alpha and beta subunits (both bind GTP) dynamic growth at both ends protofilaments align together helical shape with lumen
actin
noncovalent polymers of actin
subunits bind ATP
2 filaments twisted
flexible
intermediate filaments
rope-like made of proteins (don't bind nucleotides) (e.g. nuclear lamins/epithelial keratins) form spherical shape of nucleus alpha-helical monomers in coiled coil in cytosol
types of cell junctions in vertebrates
anchoring
occluding
channel-forming
anchoring junctions
cell-cell (adherens and desmosomes)
cell-matrix (focal adhesions)
transmembrane proteins link to cytoskeleton and to outside of cell so bind inside to outside
adheren junctions
cell-cell
link actin cytoskeletons of neighbouring cells
dimeric interactions
by transmembrane cell adhesion molecules (CAMs)
CAMs
cell-adhesion molecules cadherin family homodimer (2 same proteins) extracellular part folded into 5 cadherin repeats calcium binding sites between repeats
cadherins
secretory proteins because come form ER
stop at plasma membrane because have transmembrane domain
cadherin binding
same type cadherin in plasma membrane of cells will interact weakly
if lots bind = strong
means segregation of cells so tissue assembly and repair
diff cell types = diff cadherins = clump together to form structure
adheren vs cadherin
adheren junctions require membrane proteins called cadherins
adheren junctions also…
control motility of neighbouring cells
indirectly link actin cytoskeletons of adjacent cells via anchor/adaptor proteins
adheren in epithelial
form continuous belt
single linear band of actin connected across tissue
can tighten to cause invaginations/tubulation
remodel by pulling on cytoskeleton, epithelial tube pinches off
focal adhesions
cell-matrix
ECM to actin cytoskeleton of cell
integrins
transmembrane protein
allows internal cytoskeleton to grip onto ECM molecules (cell-matrix)
alpha/beta heterodimer
links ECM molecules to talin which links to actin
diff alpha/beta chains dinstinct ligand binding properties
low affinity binding but lots make it strong
binding to ECM and release of ECM molecules (for spreading and migrating)
switch active to inactive (bind to ECM or not) by changing conformation at both ends
other integrin functions
activate intracellular signalling pathways
why cell-matrix important?
need ECM to grow and proliferate
may die without
anchorage dependence mediated by integrins
occluding (tight) junctions function
relevant to epithelial cells (polarised cells)
so top can’t communicate with bottom of cell
block mixing of apical and basolateral membrane proteins to maintain cell polarity
stop leakage between cells
so keep 1 directional flow of nutrients
tight junctions structure
thin bands of integral plasma membrane proteins (claudin/occludin) encircle cell and these from neighbouring cells interlock
form tight extracellular seal, no diffusion across
gap junctions
channel proteins (connexons made from 6 connexins to form ring) from adjacent plasma membranes align to create channels between cells 12 subunits through both bilayers
role of gap junctions
smooth out conc gradients
co-ordinate cell responses across tissue
communicate with all other cells
functional units (tissues) should have..
structural integrity (mechanical contact)
recieve/respond to stimuli
cooperative behaviour: complexity, specialisation, organisation
how do organisms start off simple and end up complex?
selective gene expression determines 4 essential processes of development: cell proliferation cell specialisation interaction cell movement and migration
sequence of events in basic body organisation
egg
cleavage
gastrulation (lays down body axis)
germ layers (start of tissue differentiation)
proteins important for multicellular development
cell adhesion and signalling transmembrane proteins
gene regulatory proteins
desmosome junctions
intermediate filaments via cadherin proteins
what leads to variation in body plan/shape/structure?
differences in DNA regulatory proteins (transcription factors TF) and non-coding regulatory DNA (enhancers)
TF binds to enhancer region and transcription of protein occurs, which binds to downstream gene in enhancer region and makes another protein
TF can bind to a diff enhancer and make diff protein
so variation in proteins made by diff enhancers
effect of cell-cell interaction and communication (experiment)
removal, transposition, rearrangement of embryonic cells/tissues/ grown in vitro
small area of tissue transferred and produced conjoined twins (fish)
embryo divided into small number of broad regions
become future germ layer: mesoderm, ectoderm, endoderm
cells within regions become more and more committed to their fate
complexity increases as divides and goes further down specific developmental path, differentiates
as organism grows, cells will reduce……
but increase…
in mass
in numbers
2 stages of commitement
specification: is specified when cultured in neutral env. so differentiate according to fate but can change fate if in diff env.
determination: differentiate according to fate even if in diff env
goes beyond certain point where can only become 1 cell type after that
cell memory/fate
undifferentiated tissue can be regionally determined: still turn into 1 specific type no matter where you put it e.g. toes on wing
induction
inductive signal from 1 group of cells influences developmental fate of another
certain cells get stronger conc. of morphogen/protein so affected differently by signal
drives cells with same potential to follow diff path of development
depends on location of cell because of gradients
morphogens
cell-cell communication
short or long range
cell fate can also be determined by……
asymmetrical cell division
significant molecules differently distributed or
extracellular influences cause to differentiate differently
homeotic selector genes - HOX genes
regulation of animal body plan
anterioposteria patterning
sequential zones along body axis - colinearity (order of HOX genes on chromosome is same order expressed during development)
HOX proteins are transcription factors that have homeobox domain, allows binding onto HOX genes so act on each other
triploblast
intermediate and higher organisms
vertebrates
3 germ layers
3 germ layers
endoderm
ectoderm
mesoderm
diploblasts
2 germ layers (ecto and endo)
polarity and molecular asymmetry
X.leavis egg has animal pole (ectoderm) and vegetal pole (endo)
1st division down centre and across the middle so already have different contents depending on cytoplasm taken from first cell
already maternally derived polarity before fertilisation
fertilisation triggers cortical rotation of the outer cortex leading to asymmetry of mRNA
microtubule cytoskeleton moves RNAs to create asymmetrical distribution
cleavage
1st cell divisions after cortical rotation
results in many small cells (blastomeres)
first differences in cell fate
gastrulation
after cleavage (cell division), embryo becomes hollow ball of cells (blastula)
lays down tissue germ layers and body axis
anteroposterior axis
dorsoventral axis
mediolateral axis
dorsal lip coordinates movement via morphogenic gradients
VegT regulatory protein in vegetal hemisphere directs synthesis of Xnr signal proteins - classify middle cells to become mesoderms
Wnt11 signal protein activates Wnt pathway on one side of embryo (act on another gene to switch on and make other proteins)
Wnt11 signal combines with Xnr to induce Organizer (releases diffusible antagonists of Wnt and BMP
depending on where mRNA- cells within region stimulated to produce certain types of proteins
diffusable effect
cell migration
cells spatially rearranged during gastrulation
cell shape changes via convergence or elongation - convergent extension
transient protein interactions (means short time, bind and release - proteins with receptors)
move and migrate because express certain proteins, projections on cells, binding and releasing in certain direction
neurulation
creation of brain and spinal cord
relies on cell adhesion molecules
gives ability for cells to move - motility
diff expression of cell adhesion molecules in diff cell types gives tissue specificity
notochord (ectoderm) creates neural tube
somites- vertebrates, ribs, muscle
notochord (mesoderm) undergoes extension convergence - stretches out organism
neural tube (ectoderm), neural crest (ectoderm)
neural plate thicken and curl into neural tube
form brain and spinal cord (ectoderms)
limb bud
embryonic connective tissue with Sonic Hedgehog protein expression tagged blue
Sonic Hedgehog
morphogenic role
creates gradient
strongest at bottom of limb
as gradient changes from bottom up, signals formation of digits - specific orientation
chemical modifications in chromatin structure - 3 mechanisms
methylation of DNA - increase in methyl group on cytosine (can repress transcription)
acetylation of histones - become more relaxed so increase transcription
miRNAs also play a role (silencing translation)
plant development
embryo proper - dense cytoplasm
suspensor - transports nutrients to embryo
plant morphogenesis - 3 phases
differentiation
growth
division
root cap
protects meristem
modular construction
nodes maintain meristematic properties
oocyte
structure
female egg cell
outside layer = zona pellucida (layer of glycoprotein that’s species specific) - only sperm of particular species can penetrate
egg developing in follicles of ovary
1 follicle becomes dominant and ovulates
oocyte out of follicle
morula
8 cells +
blastocyst
hollow ball, whole structure
outer zona pellucida
inner trophoblast - penetrate into maternal tissue to form placenta
inside is ICM - becomes embryo, develop into hypoblast and epiblast, hatches out zona pellucida
pluripotent
embryonic stem cells
differences in placentas
diffuse placenta - pig and horse
discoid placenta - oval, primates
zonary placenta - dog
cotyledonary placenta - ruminants (cow, sheep)
epithelial tissue
tightly packed continuous sheets, ordered like bricks
polarised top and bottom, closely associated via cell junctions, separated by intercellular space
bottom basal layer anchored to basement membrane (BM)
lateral - express tight-junctions/gap, cell-cell communication
apical - top
BM 2 layers: basal lamina, reticular lamina
2 types of epithelial tissue
covering and lining
glandular
functions of epithelial tissue
protection - waterproof, minimise env. influence
selective barriers - controlled movement of substances, compartmentalise
filtration
secretion - like endocrine organ
absorption
excretion
epithelial cell shapes
squamous: squashed, flat, blood vessels
cuboidal: may have cilia or microvili, ovary, kidney tubules
columnar: may have cilia or microvili, lining GIT
epithelial cell types
simple: 1 layer
pseudostratified: 1 layer but appears like several
stratified: above 2 layers
peritoneum
lines outside of organs, simple squamous epithelium, no gaps, single layer on BM
connective tissue functions
bind support strength protect insulate compartmentalise
connective tissue
not on body surfaces, innervated, vascular
2 main elements: ECM, cells widely spaced
derived from embryonic mesenchymal cells
have stem cells: loose and dense CT have fibroblasts, cartilage has chondroblassts, bone has osteoblasts
other cells: macrophages, plasma, mast, adipocytes, leucocytes
mature CT types
loose CT - areolar, adipose, reticular dense CT - regular, irregular, elastic cartilage - hyaline, fibrocartilage, elastic bone liquid - blood tissue, lymph
muscle tissue types
skeletal
smooth
cardiac
myocytes
muscle fibres
muscle tissue functions
movement and locomotion (skeletal)
maintenance of posture (skeletal)
movement of substances (skeletal, smooth)
thermogenesis (skeletal)
muscle tissue properties
electrical excitability
contractility
extensibility
elasticity
skeletal muscle
long cylindrical fusion of myoblasts so multinucleated number of muscle fibres is set closely associated with capillary fast contraction
smooth muscle
shorter tapered at each end non striated centrally located oval nucleus stretch and recoil some myogenic
2 types - visceral (skin,tubular,ANS,gap junctions)
multiunit (lung,arteries,ANS,not as close, fewer gap junctions)
cardiac muscle
branched long fibres desmosomes intercalated discs gap junctions for coordinated contraction myogenic pacemaker conduction system mitochondria
nervous tissue
function: sensory, integrative motor
neurons (AP info, long, body dendrite axon) - unipolar, bipolar, pyramidal, multipolar (no. processes off cell body)
neuroglia - not involved in AP, smaller, supporting role, astrocytes, oligodendrocytes, microglia, ependymal, Schwann, satellite
order of tissue’s ability to regenerate
epithelial
connective
muscle
nerve
stem cells
homeostasis and repair
magnified proliferation in response to tissue damage
stem cell niche
microenvironment regulates STC fate
local env. regulates system
types of stem cells
totipotent (generate all types) e.g. zygote
pluripotent (most cell types) e.g. embryonic stem cells
multipotent (limited range) e.g. HSC
oligopotent > unipotent (few or 1) e.g. epidermal
liver has no _______ but still ________
stem cells
regenerates
integumentary system (e.g. skin)
thermoregulate
regenerate
structure of skin
epidermis (stratified epithelial and pigment cell and dendritic cells)
papillary region —-dermis—-> reticular region :loose to dense
hypodermis: fat cells, where vasculature penetrates in
other: glands, muscle, nerves, receptors, capillary loops f papilalary plexus, sweat glands, subcutaneous vasculature
fibroblast
stem cell
stem cells in skin
half way down hair follicle in stem cell buldge
migrate down: divide and differentiate into hair cell
migrate up: to sebaceous gland, replenish epithelial cells
skin layer
basal lamina (basement membrane) under epithelial some basal stem cells for epithelium, basal cell layer move away from BM differentiate into prickle cell layer, then lose organelles and pigmented = granular cell layer (dying) then kertinised squames - flake away
symmetrical and asymmetrical division
stem cell divides and 1 remains stem cell, 1 daughter differentiates to skin cell so maintain stem cells
env. asymmetry or divisional asymmetry determines which cell differentiates
transit amplifying cells
committed to differentiation
divide rapidly
programmed to specific number of amplification stages so why there are diff sized organs
identifying location of stem cells
cells that don’t have potential and stay stem cells have higher β1 integrin protein that attaches to basal layer (BM) by adhesion, stays in niche
those that aren’t bound lose stem cell properties
clusters found in basal layer
organ growth control
growth size laid down during embryonic growth via short-range signals
size determined by division, growth, death
extracellular signals
always grow to same size (transplant small dog liver to big dog)
cell turnover regulated by molecular signals
process of producing new skin cells
EGF
FGF
Wnt
EGF
epidermal growth factor
stimulates cell growth and differentiation
FGF
fibroblast growth factor
proliferation and differentiation
Wnt
series of proteins passing signals from cell surface receptors to nucleus - gene expression and cell-cell communication
vascularisation
need blood supply for growth
basal lamina of existing blood vessels break down
endothelial cells migrate to interstitial space
endothelial cells proliferate
lumen develops and matures
vessel stabilised by pericyte recruitment
endothelial cell generate new capillary branch
capillary sprout grows into surrounding tissue, hollows out to tube
tip cell has diff gene expression doesn’t divide, sends out filopodia, responds to env. signals via receptors for guidance molecules - vEGF (vascular EGF)
signals from surrounding tissue initiate angiogenesis: induces HIF1 alpha stimulates transcription of Vegf - stimulate tip cells to potrude so blood and oxygen interaction with Notch signalling pathway - which cells become tip/stalk
functional component of blood vessels
endothelial cells
vasculogenesis
early embryonic endothelial cells from 1st primary blood vessels
angiogenesis
from vasculogenesis blood vessels, elaborate network of branching to finer vessels
new capillaries from pre-existing
