• Cellular organelles. Trafficking overview.
• Intracellular membrane systems,
lysosome, peroxisome, endoplasmic
reticulum.
• The Golgi complex, endo- and exocytosis,
protein sorting.
Nuclear translocation of NF-AT
•
•
Lecture 4:
Lecture 5,6:
Dr. Bacsó, Zsolt
Dr. Nagy, Péter
•
ECB3 Chapter 15: pp 495-502, 505-530.
Membrane-enclosed organelles
•
Chemical reactions organized in cells
– e.g.: Anabolic against catabolic reactions
•
1. Ordered reactions in enzyme complexes
2. Compartmentalization
2.
1.
Outside:
Enzyme
reaction #2
Enzyme2
Enzyme1
Substrate1
Enzyme3
Substrate2
Substrate1
Substrate2
Substrate3
Substrate4
Cell ultra-structure by
Electron Microscopy (EM)
• Limit of resolution of the light microscope:
0.2 µm = 2000 Å = 2.10-7m
• Limit of resolution of the electron microscope:
in theory: 0.002 nm = 0.02 Å = 2.10-12m
at best: 0.2 nm = 2 Å = 2.10-10m
Ways:
Inside:
Enzyme
reaction #1
green fluorescent
protein GFP fused to
the gene regulatory
protein (transcription
factors) NF-AT
(Nuclear Factor
Activating T Cell)
first it is localized in
the cytosol and
excluded from the
nucleus
when cytosolic
calcium concentration
is raised by adding a
Ca ionophore, NF-AT
migrates to the
nucleus
should
exported
from
membrane
enclosed
lumen
distance between gold atoms
is about 0.2 nm (2 Å)
for biological samples:
2 nm = 20 Å = 2.10-9m
ATOMIC FORCE MICROSCOPY, SCANNING TUNNELING MICROSCOPY, X-RAY CRYSTALLOGRAPHY
Organelles:
• plasma membrane
• nucleus
• mitochondria
• endoplasmic reticulum
• Golgi apparatus
• secretory vesicles
• endosomes
• lysosomes
• peroxisomes
• cytoskeleton
•
• centrosome and centrioles
tri-laminar or “railroad” appearance of the lipid membranes in
a boar sperm cell (cytoplasm membrane at the arrowhead and
two sub-membranes at the arrows), bar - 0.05 µm.
• cytoplasm, etc.
Cytoplasm
Organelles
of the Cell
• Differentiated
structures with
maintained
composition within a
cell, that perform a
specific function.
Contains organelles, free ribosomes, cytoskeletal proteins…
Cytoskeleton
of the platelet
Cytoskeleton
branch of filaments
cytoskeleton
• Actin filaments
• Microtubules
• Intermedier filaments
Dynamic instability of microtubules
•
Microtubules
continually
grow from the
centrosome,
suddenly
however, some
microtubules
stop growing
and then shrink
back rapidly
Membrane-bounded
organelles
Relative Amounts of Membrane Types in Two Types of
Eukaryotic Cells
The Relative Volumes Occupied by the Major
Intracellular Compartments in a Liver Cell (Hepatocyte)
Percent of Total Cell Membrane
Membrane Type
Plasma membrane
Rough ER membrane
Smooth ER membrane
Golgi apparatus membrane
Mitochondria
Outer membrane
Inner membrane
Nucleus
Inner membrane
Secretory vesicle membrane
Lysosome membrane
Peroxisome membrane
Endosome membrane
Liver Hepatocyte*
2
35
16
7
Pancreatic Exocrine Cell*
5
60
<1
10
7
32
4
17
0.2
not determined
0.4
0.4
0.4
3
3
not determined
not determined
not determined
*. These two cells are of very different sizes, since the average hepatocyte has a volume of about
5000 µ m 3compared with about 1000 µm 3for the pancreatic exocrine cell. Total cell membrane
areas are estimated at about 110,000 µm 2 and 13,000 µ m 2, respectively.
Electron Microscope Tomography - Image
reconstruction from sections, cell compartments
The relationship of
late endosomes to
other membranebounded
compartments
(A) Lysosomes in Baby hamster kidney (BHK) cells
(B) Serial reconstructions of late endosomes (blue), ER (yellow), and Golgi apparatus (red)
prepared from electron micrographs, one of which is shown in (A). The reconstruction was
drawn from 18 serial thin sections. The nucleus is indicated by N in (A) and is shown in
green in (B).
Intracellular
Compartment
Percent of Total Cell
Volume
Approximate Number
per Cell*
Cytosol
54
1
Mitochondria
22
1700
Rough ER cisternae
9
1
Smooth ER cisternae plus 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 are thought to be joined to
form a single large compartment. The Golgi apparatus, in contrast, is organized into a number of
discrete sets of stacked cisternae in each cell.
Membrane-bounded
organelles
Mitochondrion
Mitochondria
•
•
Inner and outer membrane
Characteristic lamellar or tubular cristae
Oxidative phosphorylation, calcium storage, apoptosis…
Nucleus
Peroxisomes
•
Peroxisome
Smooth ER
Rough ER
•
•
Peroxisomes can be
visualized by EM since
they have electrondense crystalloid core
(urate oxidase enzyme
– but not in human!),
immuno enzyme
electron microscopy
(EM): by labeling their
specific enzyme
content (peroxisomal
oxidize, catalase)
Reactions performed:
– peroxidation
– beta-oxidation (also
in mitochondria)
– -plasmalogen
production
Glycogen
•
Detoxification:
– e.g. ethanol
•
•
•
Nuclear envelope
Nuclear pores
Chromatin
– euchromatin (bright)
– heterochromatin (dark)
•
Nucleolus
Membrane-bounded
organelles
•
Protein sorting
– Each organelles have its own protein
(lipid, carbohydrate) composition
– Proteins produced in ER and cytosol
should delivered to the right organelle
according their “mail address”
• DNA isolated from
a cell
•
Vesicular transport
– Membrane bounded small cargo vesicles
deliver some proteins like postal delivery
•
Exocytosis
– e.g. secretion, in general: materials get
out of cells
•
Endocytosis
– e.g. ingestion, in general: materials get
into the cells
Vesicular Traffic
Endocytic pathway
Endoplasmic reticulum
vesicular transport
Secretory pathway
• continuous system of interconnected tubes and
sacs (net of pipes)
– large intracellular membrane surface area
Endoplasmic
reticulum
ER dynamics
• Smooth surface ER
– Calcium stores
– Lipid, cholesterol, and
steroid synthesis
– Detoxification:
citochrom P450
hydroxylation
• Rough ER
– Ribosomes: protein
synthesis
– posttranslational
modifications
– quality control
ER dynamics and microtubuls
Microtubule cytoskeleton (red) and the membrane network of the endoplasmic
reticulum (green) dynamics at the leading edge of a migrating cell.
The endoplasmic reticulum network is
continually reorganizing.
Golgi apparatus
Localization of Specific Proteins
by EM Enzyme Cytochemistry
•
Golgi apparatus
Localization of a
particular enzyme
(nucleotide
di-phosphatase) in the
Golgi apparatus
•
(A): Golgi apparatus unstained
(B): stained with osmium in the
cisternae of the cis compartment
(C): nucleoside di-phosphatase in
the trans Golgi cisternae
(D): acid phosphatase in the trans
Golgi network.
A thin section of the cell
was incubated with a
substrate that formed an
electron-dense precipitate
upon reaction with the
enzyme.
Exocytotic pathway dynamics
Histochemical stains
demonstrate that the Golgi
apparatus is biochemically
compartmentalized
Secretory Vesicles
• Fluorescent
proteins
exiting the
Golgi
apparatus on
the way to
the cell
surface
– transport
vesicles
move along
microtubules
•
exocytosis in rat mast cells
The cell in (A) has not been stimulated. The cell in (B)
has been activated to secrete its stored histamine by a
soluble extracellular ligand. Histamine-containing
secretory vesicles are dark, while those that have
released their histamine are light.
Endosome fusion
Lysosomes
pH-sensitive
fluorescent
probe endocytosed by cells
can be used to measure the
pH in endosomes and
lysosomes. The pH in
lysosomes (red) is about 5,
while the pH in various types
of endosomes (blue and
green) ranges from 5.5 to
6.5.
• Rab5
Lysosomes
• Histochemical visualization
of lysosomes
Electron micrographs of two
sections of a cell stained to reveal
the location of acid phosphatase, a
marker enzyme for lysosomes.
– primary
(small homogenious on EM,
contains enzymes - red arrows)
– secondary lysosomes
(heterogenious on EM,
containes enzymes and
materials under digestion)