Intercellular Communication via Gap Junctions
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| License | CC Attribution - NonCommercial - NoDerivatives 3.0 Germany: You are free to use, copy, distribute and transmit the work or content in unchanged form for any legal and non-commercial purpose as long as the work is attributed to the author in the manner specified by the author or licensor. | |
| Identifiers | 10.3203/IWF/C-1624eng (DOI) | |
| IWF Signature | C 1624 | |
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| Production Year | 1986 |
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| IWF Technical Data | Film, 16 mm, LT, 113 m ; F, 10 1/2 min |
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Transcript: English(auto-generated)
00:00
Intercellular communication via gap junctions.
00:20
Cells are the components of tissues and organs. How do they exchange information which is required for the coordination of vital functions? This is a culture of synchronously beating heart cells. Frequency and amplitude of their rhythmical contractions are regulated by an ionic current
00:44
spreading directly from cell to cell via minute channels. These intercellular channels aggregate to characteristic plaques in the plasma membranes and are called gap junctions. Plasma membranes of animal cells are lipid bilayers in which proteins form a fluid mosaic.
01:08
Subunits of channels belong to these proteins. They may bind to each other and thus form a closed gap junction hemichannel, the connexon.
01:23
This extracellular part binds to a connexon of an adjacent cell resulting in a stable intercellular connection and a three nanometer gap between the lipid bilayers. More and more connexons are linked together and connect the cells in a press fastener
01:41
mode. A functional gap junction here in an oblique view contains up to several thousand connexons. Now the channels open and allow the passage of signals from one cell to another, however they do not open simultaneously but alternate independently between closed and different
02:04
open states. Thus not only the flux of the signal can be varied but also a selected transfer of information is made possible as is indicated by the different size of the passing molecules.
02:20
The maximal bore size of a connexon is about 1.5 nanometers, thus particles of a molecular weight of up to 900 Dalton can be exchanged. The inner hole of the connexons cannot be seen under the electron microscope. Their arrangement however becomes obvious after freeze fracturing of the cells under
02:42
high vacuum. As the lipid bilayer has the effect of a preset breaking line, the fracture almost invariably occurs within the cell membranes and exposes the inner membrane leaflets. The connexons stick to the plasma face of the membrane whereas the external face is
03:02
characterized by pits left by the extracted connexons. By shadowing with evaporated platinum these structures are displayed as a relief. This is stabilized by coating with a carbon film.
03:24
In anhydride of chromic acid the cellular material is dissolved. Finally the platinum carbon replica remains and can be portrayed in an electron microscope.
03:42
This picture of a replicated gap junction shows on its left the plasma face of a membrane with connexons and on its right an adjacent cell with its external membrane face and pits of the extracted connexons. Such intercellular junctions are not only demonstrated by electron microscopical but
04:03
also by electrophysiological methods. Glass micropipettes are used here. These are fabricated out of one millimeter glass capillaries which are slowly softened in a heating coil and quickly pulled apart.
04:25
In this culture of mammary tumor cells of the Marshall rat, open gap junctions will be demonstrated. Under phase contrast a micropipette filled with a fluorescent dye is inserted into a cell of the monolayer.
04:45
After switching to ultraviolet illumination only the micropipette is visible. The injection of dye into the impaled cell is performed by a negative current pulse.
05:01
After finishing the injection and withdrawing the micropipette the fluorescence still spreads into neighboring cells. The dye used here is lucifer yellow which has a molecular weight of 457 and therefore rapidly diffuses via gap junctions into other cells of the monolayer.
05:26
The nuclei always fluoresce brighter than the cytoplasm. This is elucidated by resuming the phase contrast illumination which furthermore reminds one of the arrangement of the cells.
05:46
The same experiment is now performed with a culture of epitheloid growing HeLa cells. These cells originate from a human cervix carcinoma.
06:09
Then the cell is injected with lucifer yellow for about 10 seconds and the micropipette withdrawn. In contrast to the mammary tumor cells the dye does not spread into other cells regardless
06:25
of how long the situation is observed. The absence of a direct intercellular communication is verified by electron microscopical investigations which showed the lack of gap junctions between HeLa cells. Resuming the phase contrast illumination reminds one of the close contact between these HeLa
06:44
cells which however must not necessarily be accompanied by the formation of intercellular connexons. The degree of coupling can be measured quantitatively by injecting an artificial electrical signal into the cells.
07:01
For this purpose an electrolyte-filled micropipette, a so-called microelectrode, is inserted under microscopical control. Mechanically or electrically controlled micromanipulators facilitate a precise steering of the electrodes.
07:21
Here the one for the current injection is already placed in a cell of the mammary tumor. Two others are inserted in the same and in an adjacent cell for measurements of the membrane potentials.
07:42
The result can be registered by different recording devices, for instance by a strip chart recorder where the membrane potentials with the superimposed responses of the cells are registered as well as the injected current pulses.
08:02
Pulses of milliseconds duration are observed with an oscilloscope. The two upper traces are displaced proportionally to the membrane potentials of the impaled cells. The lower trace now gives a replica of the injected rectangular current pulses and the
08:21
response of the two cells is shown together with their membrane potentials. Experiments with normal and mutant cells revealed an intercellular exchange of metabolites and nucleotides via gap junctions. Furthermore, it has been found that not only for the embryonic development but also for
08:48
the malignant degeneration of cells the direct intercellular communication is involved. The nature of the signals which are transferred from cell to cell is known only in a few systems,
09:03
for instance in the heart cells which were presented at the start of this film. The function of gap junctions and their regulation of the intercellular signal transfer is therefore studied mainly with exogenously generated signals as is shown here again for Lucifer
09:21
yellow.