bio230 2nd half

these are basically my class notes

Lec 1-3: How are cells and tissues organized spatially?

1: Membrane Trafficking

Cell Polarization

Some Cells

Polar=different at ends

Different functions based on cell regions (signal, separate, etc)

Ex. Epithelial cell basolateral domain vs apical domain

Membrane trafficking

Moving stuff to different membranes, determines where proteins end up

Eg. Move proteins to different polar domains

2 ways:

1. Exocytosis to target domain

2. Exocytosis to any domain then selective endocytosis/ recycling to target domain

Sorting stations

Organizes proteins during membrane trafficking

2 main sorting stations:

1. Trans golgi netwrok

2. Endosome

Secretion pathways (3)

• Constitutive

• Regulated (signal mediated)

• Lysosomal (signal mediated)

Constitutive secretion

• Default pathway

• Specific signals not required

• Trans golgi network —> vesicle —> membrane

• TM proteins present

• Adds phospholipid

Regulated secretion

• Release material in response to a signal

• Fully formed vesicles that dont fuse to a membrane unless signal is present

• TM protein also present

• Eg. Mast cell releases stored histamine in allergic reactions

• Regulation can give a boost of phospholipid to PM, eg after loss of membrane in phagocytosis, wound, or cytokinesis

How can Concentrated cargo occur?

• Clathrin coated vesicle buds off cargo vesicle to shrink it and goes back to golgi with just fluid

• Both constitutive and regulated can release conc. cargo

Endocytosed proteins (membrane trafficking)

membrane trafficking option 2 : exocytosis all proteins, then selective endocytosis of some of those proteins to another domain (can add to polarity)

3 options:

1. Recycle to same domain: protein binds to receptor →early lysosome → recycled BACK to og/same domain

2. Transcytosis: moved to other side/domain of PM; protein binds to receptor →early endosome→transport vesicle→recycling endosome →transport vesicle→other membrane

3. Degradation : in the lysosome

example: cholesterol uptake

Cholesterol Uptake (Ex. of Endocytosis in Membrane Trafficking)

process overview:

Low Density Lipoprotein (LDL) binds to cholesterol → LDL-C bound to PM receptor →clathrin coat vesicle forms (clathrin coat selects cargo, gives curvature to vesicle, promotes vesicle budding)→unncoating→fusion (to early endosome)

then, s basic principles

1. recycling of receptor

2. transcytosis of free cholesterol

3. degradation of cholestorol in late endosome, endolysosome, lysosome, 

3 types of membrane changes (during vesicle tracking)

1. (endocytosis) vesicles forming from donor membrane into cytoplasm
   *(outside of cell or lumen of organelle)
   eg. COPI/COPII vesicles going to/from ER & Golgi
   

2. (exocytosis) vesicle fusion: vesicle merges with target membrane
   *(outside of cell or lumen of organelle)
   eg. SNARE proteins (the pullers). Both t-SNAREs & v-SNAREs; opposite membranes to fuse.
   

3. Vesicle forms from donor membrane away from cytoplasm
   *(eg virus leaves to outside of cell and may take a bit of PM with it; may carry RNA to communicate with other cells)
   * ESCRT proteins form vesicles using machinery in cytoplasm (see image).

• 3 is the only one leaving the cell with part of the PM (net loss of PM in cell)

ESCRT proteins (cont.)

From membrane change #3 (the new, virus one that takes PM with it using machinery in the cytoplasm )

many ESCRT proteins (0-3) form vesicles, passing molecules down :

ESCRT-0 activated by a) PI(3)P and b) multiubiquitinated TM viral protein on PM

passes off a chain of ESCRT-1-3.

ESCRT-3 builds up around it and will form the vesicle

Lipid Changes During Membrane Trafficking

phospoinositides (PIPs) are found at different subcellular locations

different domains/compartments contain diff. lipids

PIPs

• phospoinositides

• part of machinery in membrane change #3

• inosital sugar(**6Cs, phosphate attached to C1, count CCW; possible additional phosphorylation sites), structural phosphate (alw there), glycerol, phosphatidylinositol (PI)

• 2 phosphorylations on the inosital sugar at C3 and C4 : PI(3,4)P₂

• interconverted by kinases and phosphatases (not every kinase/phosphatase exists)

• can use map

• different proteins bind to different PIPs which directs membrane trafficking

Rab-GTP

• molecular switches found on vesicle or target membranes

• help with vesicle formation, “matching” so correct vesicle gets brought where it needs to go

• together with PIP, can give membranes diff identities

Rab 5

1. rab 5 gtp recruits PI3 kinase

2. pi(3)p can recruit rab5-gef

3. make more active rab5-gtp

4. pi3p and rab5gtp activate tethering proteins
   
   *GDI (gdp dissociation inhibitor) bound to inactive rab5gdp

2: Cytoskeletal Networks

Polar Cytoskeleton Organization

Polar…

• microtubules: transport vesicles and proteins to ends of cell

• actin: define cell shape and behaviour

• intermediate filament: contribute to cell polarity

Polar microtubules

• transport vesicles and proteins to ends of cell

• eg. + end, - end

Polar actin

• define cell shape and behaviour

• eg. in microvilli at the ends

Polar intermediate filament

also contribute to cell polarity (more detail not req.)

Cytoskeleton dynamic rearrangments

Growth and Shrinkage

microtubules

• dimers made of monomeric proteins alpha tubulin (- end) and beta tubulin (+ end)
  *+,- ends are convention, not charge

• tubulins bind and hydrolyze GTP

• tubulin heterodimers assemble head to tail to make polarized protofilaments (alternating top to bottom)

• 13 parallel (crossection = all beta or all alpha) protofilaments form a hollow microtubule

• after being protofilament for a while beta tubulin will cut GTP to GDP

T form heterodimers vs D form heterodimers

t form: alpha and beta tubulin bound to GTP
*t form ends usually means growth/addition

d form: alpha GTP but beta tubulin bound to GDP

*d form ends usually means shrinkage/subtraction

GTP cap (dynamic instability)

• everything starts as T form; over time cut to D form- the freshest added (the cap) is still T form

• when microtubule (beta tubulin, +) is in T form and in growing/addition and the cap is not yet hydrolyzed to GDP (→ D form)

• dynamic, time sensitive

• after a while GTP → GDP

• if ends in D form, rapid shrinkage  

gamma tubulin

what about other end of microtubule (shrinkage?)?

gamma tubulin nucleates alpha ( - ) end and protects from depolymerization

growth radiates from gamma tubulin end (- end)

remains even if d form shrinkage

patterns of nucleation create microtubule patterns

how do patterns of nucleation create microtubule patterns?

fibroblast:

microtubules radiate away from centrosome (2 centrioles), surrounded by pericentriolar material (pcm) which gamma tubulin ring complexes are found

plant cell:

gamma tubulin is found on other microtubules

“branching” or seeding from another microtubule (augmin connector)

MAPs

microtubule associated proteins

kinesins “walk” towards the plus end generally

dyneins move towards minus end generally

both can hold onto vesicles/organelles with other domains and use ATP hydrolysis

Tilapia using MAPs motor proteins

microtubule motor proteins can change fish color

Kinesins walk to + end; dyneins walk to - end

In dark fish they both carry vesicles with pigment and compete with each other; even distribution of pigment.

In light fish kinesins are inhibited, pigment is no longer moved outwards and tightly aggregates in the cell centre; light pht is observed.

polar actin filaments

• made of monomers that are asymmetric and thus “polar”

• can bind and hydrolyze ATP

• by convention (not charge), - end bottom, + end top

• 2 monomer strands assemble (twizzler style) into polarized actin filaments

• plus end literally ends with a +monomer and vice versa

• T form monomers (actin ATP) and D form monomers (actin ADP)
  *same basic concept as microtubule time sensitive GTP→GDP bound

Treadmilling

actin filaments soluble subunits in tform

polymers are a mixture of tform and dform(old)

at the right conc. of actin: some addition of actin at + end but not at the  minus end (concurrent loss at - end, gain at + end)

ARP2/3 complex

• actin related proteins

• helps nucleate actin filaments (stabilize - end of actin and protect from depolymerization)

• equivalent of gamma tubulin in microtubule?

• uniquely nucleates actin filaments on pre-existing filaments (branched, seeding thing)

• creates polar network of actin in cell (generally + towards membrane, - inside)

NPF

nucleation promoting factor

• NPF binds to inactive Arp2/3 complex

• t form actin assembles on other plus end

Ways actin can move (4)

elongate

shrink

treadmill

stabilize with arp2/3

Cell crawling (how can a cell move with actin?)

• uses polar actin network

• actin getting added to leading edge (membrane region of cell)

• new branches from arp2/3 poking the membrane forward

• actin treadmilling (poke forward while loss on other end)

basically: poke poke poke, other side contractile & getting dragged along

cofilin

• chopping protein

• cuts actin filament; exposed d form actin gets degraded quickly

• release from arp2/3 to help deassemble actin

• found in network treadmilling (where + end poking)

Integrins

• TM heterodimers (alpha, beta subunits)

• anchor actin to extracellular matrix (ECM) proteins

• indirectly interact with actin

• provide adhesion necessary for cell migration

• other proteins help w this too

myosin

motor proteins; motor domains use atp for motor domains

works with actin to generate force (cell migration, muscle contraction)

Rho family small GTPases

• influence actin organization → cell shape, polarity and behaviour

• act as molecular switches

• RhoGAP and RhoGEF, GTP binding and hydrolysis (similar concept to RanGTP in nuclear transport)

Rho family GTPases members? (3)

• Family members: Rho, Rac, Cdc42 proteins

  • overactivation of any members leads to varying actin organization patterns (next slides)

Quiescent cells

normal Rho family GTPase activation (normal actin behaviour, cell movement), healthy balance b/w members

cell in interphase

Cdc42 overactivation

• Cdc42 overactivation (lots of Cdc42 gef) → spikes

Rac overactivation

• Leading edge around the entire cell

• not normal

Rho overactivation

myosin contraction throughout the cell (as opposed to only the dragged behind end)

Rac GTP activation

responsible for leading edge “poking” behaviour

in a normal cell Rac GTP activation occur mainly at front of cell

Rho GTP activation

responsible for contractile behaviour

in a normal cell, Rho GTP activation occurs at “back” of the cell

(eg. this neutrophil chase)

chemoattractant

bacterial waste(?) that notifies a neutrophil (bacteria eater cell)

that activates receptor for Rac GTP to form leading edge

cell polarity in fertilized egg/zygote

sperm (posterior) enters egg ; symmetry breaking occurs

cytoskeleton changes to polar

bigger theme of blob →distinct phenotype

3: Cell Adhesion

cell cohesion

types of tissue work tgt (eg.?)

essential for multicellular organisms

epithelial cells

• line surfaces, cavities, and organs

• protective (skin)

• absorption (digestive tract)

  • define (organs)

• polarized: apical, basal

epithelial structure

Apical (top)

basal (bottom; facing basal lamina)

require junctional complexes

epithelial junctions (6)

apical

• tight: seal gap bw ep cells, block, keep stuff on right side, form sealing strands
  
  cell-cell anchoring junctions:

• adherens: deeper into plane, 3d adhesion, form adhesion belt, anchors actin bundles of two cells O-O

• desmosome: connects int. filaments, anchors int. fil of two cells

• gap: lets small soluble (water) thru bw cells

• actin linked cell matrix ALCM: sticking cell to ECM via actin + ECM proteins

• hemidesmosome : anchors int fil to ECM
  *these last two are next to each other in the bottom

basal

epithelial junction (category)

apical

• tight junction

  • tight

• cell-cell anchoring junctions (2)

  • adherens

  • desmosome

• channel forming junction

  • gap

• cell matrix anchoring junctions (2)

  • ALCM

  • hemidesmosome

basal

how do cell-cell junctions work?

mediated by cadherin family members

use TM adhesion proteins (integrins) to stick two cells

actin or int. fil.s

cadherin family members

• @ adherens junctions

• TM proteins expressed by both cells, stick to each other outside of cell

• like velcro

• homophilic intc.s (only the same types of cadherins stick to each other)

• req. Ca2+ to stick to ea other (keeps it rigid)

homophilic cadherin interactions

• allow cells to sort into groups (like the hydrophobic effect)

• cells with E-cahderins; cells with N-cadherins

adhesion belts

• formed by adherens junctions (which anchor actin bundles of 2 cells)

• can mediate morphogenesis, embryogenesis

• contraction can form tube structures (eg formation of spinal chord)

apical domain ep cell

faces surface, cavity, organ

basal domain

faces inside of the body

basolateral domain

basal and lateral domains

tight junction

physically defines domains (apical, basal, etc)

limits diffusion from one cell to the next

occludins & claudins

monomers of tight junction (many rows make up a )

TM proteins that form homophilic interactions with their extacellular domains to directly link adjacent cells

tight junctions & glucose conc.

glucose conc. (low outside ep cell, high in centre of ep cell) maintained by transporters being on the right side (apical vs basolateral) by tight junctions

how?

glucose found in lumen of gut - food filled cavity lined with epithelial cells

apical side contains:

sodium driven (active) glucose transporters (kept on apical side by tight junctions) →high conc. glucose pumped in cell

basolateral side contains:

passive glucose transporter proteins (kept on that side by tight junctions)→glucose leaves cell (eg. thru bottom; basal lamina to blood)

glucose has to go through cell; cant passively diffuse to blood/connective tissue

sodium driven (active) glucose transporters

moves glucose from apical into ep cell

creates low to high conc grandient

passive glucose transporter proteins

allows glucose to diffuse from basolateral side to basal lamina/blood

(to adjacent cells???)

cell-matrix junctions

play roles in both epithelial and connective tissue

what order/cues of junctions are made in ep cell?

adherens junctions form first:

• help cells stick together

• provide polarity cues to define apical domain from basolateral domain

adherens junctions give polarity signals that activate PAR, Crumbs, Scribble complexes

tight junctions

landmark

can be a structure, protein, signal or process

initial landmark can generate subsequent patterns/elaboration

eg. adherens junctions is landmark; leads to cell polarity

Lec 4-6: How do multicellular organisms develop?

4: Tissue Morphogenesis

(3) key concepts in multicellular development

1) cell proliferation

2) cell differentiation

3) cell morphogenesis

cell proliferation

increase in cell numbers

cell division

(eg cleavage of fertilized egg to the blastula)

cell differentiation

change in cell fate (function, location) via cell signaling and differential genome expression

(eg. gastrulation of blastula to gastrula)

cell morphogenesis

change in cell shape, interactions and/or locations

(eg. gastrulation of blastula to gastrula)

embryogenesis

initial stages after fertilisation of the egg

model system for multicellular dev:

busy time for multicellular development + short reproducible timeline → great to study these processes

does multicellular development occur in adults?

yes, particularly in stem cells

ongoing (eg skin/ep cells) and new (eg. pregnancy) developmental changes 

embryogenesis steps

1) cleavage (many cell divisions) of fertilized egg

2) gastrulation of blastula to create gastrula

fertilized egg → (cleavage) →blastula →(gastrulation)→ gastrula

blastula

sphere of cells, hollow/ filled with fluid

created by cleavage/many cell divisions

fairly undifferentiated (can become any cell)

gastrulation

• change from ball of cells to embryo with a gut and 3 germ layers (eg. ectoderm, mesoderm, endoderm)

• formation of the gut tube (yellow) eventually forms the digestive tract

• blastula folds in on itself

• cell types begin to emerge

partial differentiation

morphogenesis

the generation of shape

(3) key concepts of morphogenesis

1. cell internalization

2. elongation

3. fine repositioning of cells

(how does gastrula esque blob acc become an animal looking thing)

1. cell internalization

delamination, ingression, involution, invagination

3 germ layers

each layer becomes different kinds of cell tissues in the adult body

ectoderm, mesoderm, endoderm

ectoderm

becomes the epidermis & nervous system

mesoderm

becomes the muscles, connective tissue, bones, blood, kidneys, etc

endoderm

becomes gut, lungs, pancreas, liver, etc

stupid TCP analogy of gastrulation/3 layers

blastula is kinda like if you have the younger group of students that could become any specialty, then once gastrulated the older kids pick a path and still could do a variety of things, but cant go back and do anything. like endoderm cells can become pancreas, gut, lungs but they cant go back and become NS or skin. its like choosing medicine pathway and you can be a nurse, researcher, surgeon but not a graphic deisgner or some shit idk. making me think abt the academy and its reach. how many things does it do? what does it train its kids to become? is it monopolizing anything? roots deep in the cities etc. anyway. back to lec

(2) process to form mesoderm

ingression and delamination

ingression

process to form mesoderm

individiual cells detach from the outer cell layer and migrate (green, cells lost adhesions)

aka epithelial-to-mesenchymal transition

delamination

process to form mesoderm

cells forming a new layer (mesoderm)

epithelial-to-mesenchymal transition

ingression

cell changed from epithelial cell (tighly adhered to neighbors) to crawling type cell (mesenchymal cell)

tightly controlled; potential for malignant cell growth

(2) processes to make endoderm

invagination

involution

invagination

• form endoderm

• sheet of tight cells curls in to form pouch/cavity

  • elongation of mictrotubules in sheet of ep cells → invagination by adhesion belt tightening in select areas
    *req actin, cell adhesion

involution

curling in of the cells

gut tube

• one end develops to become the mouth, the other end develops to become the anus 

• forms from endoderm

vertebrate neural tube

• forms similar to gut tube (invagination/involution)

• ectoderm → neural plate cells → neural crest plate →neural tube

• relies on dev signalling, diff. gene expression (eg. of cadherin)

2. elongation

congergent extension, mass cell migration, asymmetric growth

convergent extension

• involves cell movement

• cells come together (converge) and line up (extend as a tissue)

• same number of cells throughout; organiszation differs

• after gastrulation