* unanswered questions .. debates

Cornelius Krasel krasel at wpxx02.toxi.uni-wuerzburg.de
Sat Jul 6 05:37:01 EST 1996


Raeto West (101722.35 at CompuServe.COM) wrote (for Harold Hillman):
> On 26 Jun 1996, Margaret Fowler wrote:
> > Ricardo Azpiroz <azpiroz at U.Arizona.EDU> wrote:
> > >3) Read about axonal transport of synaptic vesicles. There are VIDEOS
> > >showing vesicles moving down microfilaments. Movement and cytoskeleton,
> > >right before your eyes.
>
> > 3) Synaptic vesicles can only be seen by electron microscopy of *dead
> > tissues* in which intracellular movements do not occur. Therefore the
> > movement of *vesicles* can not be seen. Sheetz' beautiful videos do not
> > show mitochondria being pulled along. His microtubules can be seen by
> > light microscopy (with a resolution of 200-250 nm under *best*
> > conditions), but microtubules (see Dustin Amos and others) are < 25 nm
> > and are therefore not the same structures.

You should learn about video-enhanced light microscopy before
stating the obviously wrong.

[Warning: change of attributions follows!]

Raeto West (101722.35 at CompuServe.COM) wrote (for Harold Hillman):
> cytoana at univ-lyon1.fr (Richard DELORME) wrote:
> >In article Margaret Fowler <101722.35 at CompuServe.COM> writes:
>
> >>    (b) That the following structures do not exist in the living
> >>cells: endoplasmic reticula, Golgi bodies, lysosomes, nuclear pores,
> >>mitochondrial cristae, the cytoskeleton, actin filaments and synapt-
> >>ic knobs, either because they would not permit the evident intra-
> >>cellular movements, or because they disobey the laws of solid
> >>geometry. Transmembrane molecules and receptors can not be seen on
> >>the cell membranes by transmission electron microscopy, although
> >>sequencing shows them to be 2-3 times the diameter of the cell
> >>membrane, which *can* be seen by electron microscopy;
>
> >It is possible to see some of this structure IN the living cells. For example,
> >DIOC6 is a fluorescent dye that stains the endoplasmic reticula in the living
> >cell, rhodamine 128 stains mitochondria, etc... If you are a web surfer, you
> >can find some movies showing these structures moving inside the cells.
>
> Nearly all fluorescence, if not all, is done in the fixed, dehydrated and mounted
> sections in which movements can not occur. Do you do immuno-fluorescence?

Mr. Delorme wrote about staining of *living* cells, not about
immunofluorescence. This is at least the third time that I have
seen either in private email or in public debate that you avoid
evidence if you don't like it. (For the record: one time was
when I pointed out that topographical mapping of beta-adrenergic
receptors has been done on living cells as well as on membrane
preparations and on dead cells; the second time was the complete
ignoring of a private email pointing out that there is no difference
between ligands and agonists, that naturally occuring ligands are
used in a few cases to study receptors, and that artificial ligands
are used in other cases because they are more specific. Mr. Hillman
responded to this by again posting that artificial ligands were the
only ligands being used in receptor binding studies and that this
would only result in artifacts.) If this is your idea of a discussion,
I will quickly drop out of it.

[Again change of attribution due to Mr. Hillman's unusual quoting style:]

Raeto West (101722.35 at CompuServe.COM) wrote (for Harold Hillman):
> anthonyp at scripps.edu (Anthony J. Pelletier  Ph.D.) wrote:
> >Below, you ask me to answer your 45 questions. Why won't you answer one of
> >mine? I asked you how you explain the visualization of microtubules in
> >*living* cells.
> >   I understand your concern over fixation artifacts...we all are concerned
> >with them. This is why alternate methods have been employed.
> >   So, one can micro-inject fluorescent tubulin, wait a while for the cell to
> >recover, do all the correct controls to show that, following the
> >microinjection, the cells continue to live, grow, divide etc, and
> >visualize, directly, the microtubules in the living cell. Moreover, since
> >the fluorescent dye photobleaches, you can bleach out the dye in a defined
> >region and follow the incorporation of new tubulin into the network.
> >Living cell...not dehydration artifact. Explain please?
>
> I should be grateful for references to this. However, microtubules which
> can be resolved by light microscopy (200-250 nm) are 10x diameter of those
> seen by electrons (<25 nm) therefore could not be the same.

See pictures of this in Alberts et al, Molecular Biology of the Cell,
Garland Publishing, which should be available in any cell biology
library (since it is a book for students). Again Mr. Hillman is ignoring
that a microtubule which emits light can be seen by light microscopy
although it cannot be resolved in its actual dimensions.

--Cornelius Krasel (who is neither a PhD nor a teacher).

--
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP3 */
/* "Science is the game we play with God to find out what His rules are."  */



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