1/17

Overview of compartments and intracellular transport

Material for three lectures (Cellular organelles, trafficking overview + Intracellular membrane systems, lysosome, peroxisome, ER + Golgi,

endo- and exocytosis, protein sorting):ECB4 Chapter 15: pp 487-494, 498-523.

Keywords for the three lectures: compartmentalization, endosymbiotic theory, vesicular transport, detoxification, smooth ER, rough ER, Golgi

complex, peroxisome, lysosome, acid hydrolase, storage disease, autophagy, quality control in the ER, cotranslational translocation, ER

signal sequence, glycosylation, protein sorting, SNARE proteins, endocytosis, exocytosis, phagocytosis, transcytosis, receptor recycling,

regulated secretion, constitutive secretion, receptor-mediated endocytosis, clathrin, familial hypercholesterinaemia.

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Lecture objective

Have you ever wondered • why our cells have so many organelles?• if our cells could manage without organelles?• if the existence of organelles is rooted in evolution?

By the end of the lecture you will understand• how compartments (organelles) are organized to form a system in a eukaryotic cell• how these compartments contribute to the survival of cells by communicating with

other compartments by• biological• physical (diffusion)means

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Studying the structure of cells and organelles by microscopy

Resolution limit of a light microscope:2000 Å=200 nm=0.2 mm=210-7 m

Resolution limit of an electron microscope:in theory: 0.02 Å=0.002 nm=210-6 mm=210-12 min practice: 2 Å=0.2 nm=210-4 mm=210-10 min biological samples: 20 Å=2 nm=210-3 mm=210-9 m

REVISION

4/17Lodish: Mol. Cell Biology, 5th edition, Fig. 1-2.

Comparison of the compartments in prokaryotic and eukaryotic cells !

5/17

Importance and realizations of compartmentalization• It mainly plays an important role in eukaryotic cells.• Biochemical reactions take place in an organized way in cells: different reactions with different

requirements at the same time.• These reactions have to take place in a spatially separated (compartmentalized) way.• Realizations:

1. „Virtual compartments” (without membranes): enzyme complexes (in eukaryotes and prokaryotes)

2. Compartmentalization (by membranes, only in eukaryotes)

Enzyme 1

Enzyme 2

Enzyme 3

substrate 1product 1=substrate 2

product 2=substrate 3

product 3

Enzyme 4

Enzyme 5

Enzyme 6

substrate 1product 4=substrate 5

product 5=substrate 6

product 6

substrate 1

product 1substrate 1

product 2

Due to compartmentalization transmembrane transport is necessary which is often energy-consuming.

product 1

!

Example for virtual compartments:

A

B

C

Synthesis of “C” by an enzyme complex: efficient since there is no need for diffusion

A

B

A

B

B

C

B

C

Diffusion of “B” is required slows down the synthesis of “C” from “A”

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Droplet organelles

• a recently discovered form of „virtual compartments”• generated by liquid-liquid phase separation (like when mixing water and oil), and they

behave like liquid droplets:o they fuse with each other

Liquid droplets formed by fluorescently labeled, multivalent binding proteins (multi-SH3 domain, multi-proline rich region) and their fusion.

o diffusion takes place inside them and they exchange material with their surroundings

• such droplet organelles are the nucleolus, Cajal bodies, etc.

Diffusion observed in a droplet organelle by FRAP.

• Hyman, A. A., Weber, C. A. & Julicher, F. Liquid-liquid phase separation in biology. Annu Rev Cell Dev Biol 30, 39-58

• Courchaine, E. M., Lu, A. & Neugebauer, K. M. Droplet organelles? EMBO J 35, 1603-1612

info

7/17

Organelles and their relationship to each other 1.

• cell nucleus• mitochondria• endoplasmic reticulum• Golgi• secretory vesicles• endosomes• lysosomes• peroxisomes

• cytoskeleton• centrosome• ribosome• nucleolus• Cajal bodies, …

Organelles: specialized structures in cells that have specific functions (less restrictive definition)

Advantage of compartmentalization:• membrane-bound organelles establish special

conditions favoring a given set of biological functions (e.g. lysosome – hydrolytic degradation, mitochondrion – ATP generation, etc.)

• different reactions with different requirements at the same time

Disadvantage of compartmentalization:• transport across compartment boundaries is complex

and often energy requiring

membrane-bounded organelles: according to the generally accepted, more restrictive definition only these are considered to be organelles, i.e. specialized structureshaving specific functionbounded by a membrane

!

8/17

Organelles and their relationship to each other 2.

Wikipedia

1. Nucleolus2. Nucleus3. Ribosomes (little dots)4. Vesicles5. Rough endoplasmic reticulum6. Golgi apparatus7. Cytoskeleton

8. Smooth endoplasmic reticulum9. Mitochondrion10. Vacuole11. Cytosol12. Lysosome13. Centriole14. Cell membrane

!

9/17

Organelles and their relationship to each other 3.

Evolution of the nucleus and the ER

Evolution of the mitochondrion

cytosol

nucleus

extracellular space

vesicleslysosome

ER, nuclear membrane

1. The extracellular space and connected compartments : EC space, ER, Golgi, lysosome, vesicles (endo- and exocytic, transport), the lumen of the nuclear membrane and the intermembrane space of mitochondria

2. Cytosol of the ancient prokaryotic cell: cytosol, nucleus

3. Engulfed cells: inner membrane and matrix of the mitochondrion, peroxisome

Three compartments from an evolutionary point of view:

mitochondrion

peroxisome (?)

Golgi

!

10/17

Organelles and their relationship to each other 4.

cytosol

extracellular space

vesicleslysosome

1. The extracellular space and connected compartments: EC space, ER, Golgi, lysosome, vesicles (endo- and exocytic, transport), the lumen of the nuclear membrane and the intermembrane space of mitochondria

2. Cytosol of the ancient prokaryotic cell: cytosol, nucleus

3. Engulfed cells: inner membrane and matrix of the mitochondrion, peroxisome

Three compartments from an evolutionary point of view:

mitochondrion

peroxisome (?)

Two compartments from the stand point of intracellular transport:

cytosol

extracellular space

vesicleslysosome

ER, nuclear membrane

mitochondrion

peroxisome

nucleus

1. endomembrane system: ER, Golgi, lysosome, vesicles (endo- and exocytic, transport) and the lumen of the nuclear membrane (+EC space, plasma membrane)2. cytoplasmic compartment:2a. cytosol

2b. organelles communicating with the cytosol by non-vesicular transport: nucleus, peroxisome and the mitochondrion

transport between individual organelles by means of vesicular transport

diffusion/directed motion + transmembrane transport

nucleus

ER, nuclear membrane

GolgiGolgi

!

11/17

Organelles and their relationship to each other 5.

cytosol

lysosome

peroxisome

diffusionspecific transport across nuclear pores

porinpore

diffusion across the porin pore up to 5000 Dalton

TIM-TOM complex

specific transport across TIM-TOM or peroxin complex

peroxin

mitochondrion

ribosome+mRNA

VESICULAR TRANSPORT

nuclear pore

The principle of transport in the cytosolic compartment (compartment 2): diffusion (random or directed) + transmembrane transport (without vesicular transport steps involved).

!

12/17

The Nobel Prize in Physiology or Medicine 2013 was awarded jointly to James E. Rothman, Randy W. Schekman and Thomas C. Südhof "for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells".

Nobel prizes awarded for discoveries related to intracellular membrane systems

James E. RothmanRandy W. Schekman Thomas C. Südhof

Using genetic screens of temperature-sensitive mutants of Saccharomyces cerevisiae he identified genes (sec genes) involved in membrane and vesicle trafficking.

He described the role of calcium in regulating exocytosis and identified synaptotagmin, the main calcium sensor in the exocytosis of neurotransmitters.

He investigated the trafficking of VSV G protein (VSV – vesicular stomatitis virus) and identified NSF, SNAP and SNARE proteins.

The Nobel Prize in Physiology or Medicine 2016 was awarded Yoshinori Ohsumi “for his discoveries of mechanisms for autophagy”.

Yoshinori Ohsumi

13/17

Diffusion in the intra- and extracellular space

2 2 2 2 6r x y z Dt 2r t

distance travelledtime of diffusion (sec)

protein glucose glycine

1 mm 0.0017 0.00033 0.00017

10 mm (size of a eukaryotic cell)

0.17 0.033 0.017

100 mm (max. distance of cells from capillaries)

16.7 3.3 1.7

1 mm 1667 333 166.7

1 cm 166667 33333 16667

Diffusion is an important means of transportation in biological systems, but it has limitations

physical limitation 1

glycine (MW=75)

glucose (MW=180)

ordinary (40 kDa) protein

D (m2/s) 10-9 510-10 10-10

D (cm2/s) 10-5 510-6 10-6

D (mm2/s) 103 5102 102

approx. 1000x difference in MW

approx. difference in D3 1000 10x

due to physical

limitation 2

!

3

1 1kTD D

f f MW

k – Boltzmann constantT – absolute temperaturef – form factorMW – molecular weight

physical limitation 2 (weak)

Based on the above• diffusion is an efficient way to transport molecules (up to 100 mm).• it is not efficient for transport to larger distances.BUT: there are other, biological limitations (see next slide) the diffusion of proteins is significantly hindered or confined in the cell

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Diffusion in the intra- and extracellular space: filtering effect of the cytoplasm

The rate of diffusion is further decreased by:• molecular weight dependent (static) filtering effect of the cytosolic matrix with a pore

size of ~50 nm • dynamic filtering effect of macromolecules (see below)• specific interaction of proteins with the cytoskeleton (or DNA in the nucleus)

bundle

meshwork

biological limitations

Finding: the diffusion of proteins declines steeply with their molecular mass large proteins (300-500 kDa) basically stop diffusing.

High concentration of dissolved macromolecules can also create a

dynamic barrier.

effect of the cytoskeleton: besides the above dynamic barrier a static fence is

also created by the cytoskeleton.

completely locked

sometimes locked

completely free

!

Intracellular transport of

proteins small molecules (e.g. glucose)

diffusion is only efficient for short-range transport (up to 1 mm)diffusion is efficient for transport throughout the cell (up to 100 mm)cytoskeleton-mediated directed transport for transport of proteins

throughout the cell

Structure behind static filtering

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Relative amounts of membrane types in two different eukaryotic cells

Percent of total membrane in the cellType of membrane Liver cell (hepatocyte)* Panreatic exocrine cell*Plasma membrane 2 5RER membrane 35 60SER membrane 16 <1Golgi apparatus 7 10Mitochondrion

Outer membrane 7 4Inner membrane 32 17

Inner membrane of the nucleus 0.2 3Secretory vesicle membrane not determined 3Lysosome membrane 0.4 not determinedPeroxisome membrane 0.4 not determinedEndosome membrane 0.4 not determined* These two cells are of very different sizes. The average hepatocyte has a volume of about 5000 mm3 compared to 1000 mm3 for the pancreatic exocrine cell. Total membrane areas are estimated at about 110000 mm2 and 13000 mm2, respectively.

!

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Relative volumes occupied by the major cellular compartments in a liver hepatocyte

Intracellular compartment

Percent of total cell volume

Approximate number per cell

Cytosol 54 1

Mitochondria 22 1700

RER 9 1

SER and Golgi cisternae(Golgi)

6 (3) 1

Nucleus 6 1

Peroxisomes 1 400

Lysosomes 1 300

Endosomes 1 200

* All the cisternae of the rough and smooth endoplasmic reticulum arethought to be joined to form a single large compartment. The Golgi apparatus,in contrast, is organized into a number of discrete sets of staked cisternae ineach cell.

!

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Summary

• There are two major compartments in a eukaryotic cell from the stand point of

transport,

• which mirror the evolutionary origin of organelles.

• Diffusion is efficient for short distances (how short? depends on the substance

to be transported),

• while directed transport (mediated by the cytoskeleton) is needed for longer

distances.