* Unanswered questions: debates *

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


Re: Unanswered Questions
Received: from kitts.u.arizona.edu (kitts.U.Arizona.EDU [])
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)
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 

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>
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' 
>	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 []) 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.


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