* Unanswered questions: debates *

Margaret Fowler 101722.35 at CompuServe.COM
Wed Jun 26 07:32:02 EST 1996


RICARDO ASPIROZ; RICHARD DELORME; RICHARD KONDO; ANTHONY PELLETIER:
DEBATES IN PROGRESS OVER DR HAROLD HILLMAN'S 'UNANSWERED QUESTIONS'

Re: Unanswered Questions
Received: from kitts.u.arizona.edu (kitts.U.Arizona.EDU [128.196.137.17])
Date: Tue, 18 Jun 1996 16:51:55 -0700 (MST)
From: Ricardo Azpiroz <azpiroz at U.Arizona.EDU>
To: Harold Hillman <101722.35 at CompuServe.COM>

>Very well, if you don't wish to friendly about this, consider the following:
>
>1) The reason I don't read Hillman is that I have never heard of
>him/her/you and the work cited. I have read dialectical critiques of
>modern science, and agree with much of them. So, I do have an open mind.

1) You should be prepared and willing to answer my questions without reference
to my publications. I was only pointing out that I have published answers.


>2) If I sent you a micrograph with what you call random orientation of a
>bilayer, you would, rightly, claim it to be uninterpretable; such
>micrographs are only interpretable in the context of serial sections.

2) *I challenge you* as I did anyone to send me an electron micrograph with
the cell, nuclear and mitochondrial membranes, the cristae, the endoplasmic
reticulum, the synapses in a random selection of orientations. *You* can
put arrows showing me and all the other Usenet users every orientation in
*one* micrograph. I repeat this challenge to any electron microscopist
in the world.


>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.

Harold Hillman.


====================================
Harold Hillman
From: cytoana at univ-lyon1.fr (Richard DELORME)
Subject: Re: * UNANSWERED QUESTIONS: RESPONSE *
Date: Fri, 14 Jun 1996

>In article <4p7dc4$t16$1 at mhade.production.compuserve.com> Margaret Fowler <101722.35 at CompuServe.COM> writes:
>>From: Harold Hillman <101722.35 at CompuServe.COM>
>>Date: 6 Jun 1996 20:00:04 GMT
>
>>    (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?


>Of course, if you are blind against any kind of evidences...

Rude. You have not read my publications, so how do you know?


===============================================
From: Richard Kondo <rkondo at ephys.ucla.edu>
Subject: Re: * UNANSWERED QUESTIONS: RESPONSE *
Date: Mon, 10 Jun 1996 13:35:21 -0700
Organization: UCLA Cardiovascular Research Lab

>Harold Hillman wrote:
>> 
>> 1. Discourtesy, assumptions of ignorance, and emotive remarks are no
>> substitute for measured argument and evidence. Each of these quest-
>> ions highlights a contradiction *within* current views; for example,
>>     (a) everyone agrees that intracellular movements can be seen by
>> low power light microscopy in living cells, yet most people also
>> believe that there is a cytoskeleton, which would not permit such
>> movements;
>
>	rest deleted
>
>	On the contrary, the cytoskeleton is most likely essential 
>for intracellular movement of organelles.  The role of microtubules, 
>associated structural proteins and the ATP driven protein motors, 
>kinesin and dynein have been elucidated over the past 15 years.
>
>	Brady et al., (1982) Science  'Fast axonal transport in 
>extruded axoplasm from giant squid axon'  218:1129-31.
>
>	Hayden et al., (1983) Cell Motility 'Cytoplasmic transport in 
>keratocytes: direct visualization of particle translocation along  
>microtubules' 3:1-19
>
>	Schnapp et al., (1985) Cell 'Single microtubules from single 
>axoplasm support bidirectional movement of organelles' 40:455-62

Schnapp et al. 1986 Ann NY 4 466 509-518

>	Vale et al., (1985) Cell 'Identification of a novel force 
>generating protein, kinesin, involved in microtubule-based motility' 
>42:39-50
>
>	Sheetz et al., (1987) Annals of New York Academy of Sciences, 
>'Movement of vesicles on microtubules' 493:409-16
>
>	Schnapp and Reese (1989) PNAS 'Dynein is the motor for 
>retrograde axonal transport of organelles' 85:1548-52.

Vale R D, Schnapp (1985) Cell 43(3) part 2 623-632

 
I am sending you my paper (Hillman, 1991) 'Some microscopic considerations
about cell structure - light versus electron microscopy' Microscopy *36*,
557-576, dealing with this question in detail.
   Meanwhile, a few remarks.
   I am sure that you have seen micrographs of networks of tubulin, vimentin,
spectrin, endoplasmic reticulum, microtrabeculae and actin. In these you do
*not* see lysosomes, Golgi bodies or mitochondria, and with *all* these
elements together, they would not allow *enough space* for relatively large
bodies to move round. The hypothesis that, for example, actin can pull
mitochondria requires a mechanism and attachments all round the mitochondria
*and to other structures* otherwise they could not pull. The maximum
resolution of the light microscope - under which intracellular movements are
seen in living cells - is 200-250 nm, yet the electron microscopists describe
them as 25nm. Therefore the microtubules are not the same structures; this
also applies to those believed to be the spindles pulling the chromosomes to
the poles during cell division. The hypothesis that the cytoskeleton pulls,
say mitochondria, ignores the possibility - a simpler one - that Brownian
movement, diffusion, streaming, convection movements (none requiring
biological mechanisms) occur in fine granules in fluid.

Harold Hillman.

=================================================
Re: Unanswered Questions
Anthony J. Pelletier  Ph.D.
Received: from scripps.edu (riscsm.scripps.edu [137.131.120.6]) by arl-img-5.compuserve.com
Date: Tue, 18 Jun 1996 16:42:23 -0700
To: Harold Hillman <101722.35 at CompuServe.COM>
From: anthonyp at scripps.edu (Anthony J. Pelletier  Ph.D.)

>>Because according to cytologists the cytoplasm has a thick skein of
>>microtubules, actin, spectrin, tubulin, vimantin, etc. These are found
>>in dead, stained dehydrated tissue. Dried out salt solution contains
>>similar strands as do snow flakes but liquid H2O does not have a
>>network. The moving particles are 10-100 x the diameters of the space
>>between the fibres. Please see Hillman and Sartory (1980) 'The Living
>>Cell'. The cytoskeleton is a precipitate of the cytoplasm resulting
>>from dehydration during preparation.
>
>I'll have to read your work to find out where you get the "10-100X" figure.
> But real-time measurment of "microdiffusion" of particles in living cells
>suggests that some of the large particles are in fact constrained for short
>periods within actin "cages" and move from place to place only when the
>actin skeleton rearranges locally to allow it. Treatment with actin
>de-polymerizing agents removes the "cage" effect.

Do you know of any electron micrographs showing mitochondria attached
to any part of the cytoskeleton in such a way?


>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.


>Also, consider the mutations in the genes encoding the proteins of the
>cytoskeleton. The effects on cell morphology are as predicted from the
>model. How does your model account for the effects of mutations to
>cytoskeletal proteins on cell shape, motility etc. Indeed, how does your
>model account for cell shape and motility at all?
>
>>Dear Dr Pelletier,
>>You are entitled to believe that I am wrong, if you have read my
>>publications. I should be pleased to supply you with reprints on
>>particular points.
>
>Absolutely send me any reprints you feel are appropriate.
>
>>I would be more pleased if you would answer the
>>45 questions.
>
>Not to be rude, but you have not yet asked a question I find compelling.
>Perhaps your reprints will help with that. And, in the cases where I
>attempted to address you questions with data, you ignored them.

You do not have to find them compelling. They are reasonable questions
to which you - as a teacher - should have answers.


>-tony





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