Monthly Archives: February 2018

Great research on desmosomes, but

Desmosomal mitochondrial associations, Ultimate order, the cell

All you guys out there that are doing great research on the molecular structure of the proteins comprising the desmosome, BUT, there is a huge disconnect in what is visible with routine electron microscopy, and the diagrams that are being produced.  Just for starters, if I (not being an engineer but just a craftsperson) were going to build a rigid structure binding two adjacent objects i would NOT make it unidirectional.  That is I would not orient all the layers of the molecules in parallel.  I would do what is commonly done in ply wood, that is to plait the structure with perpendicular, parallel, perpendicular, parallel layers.

The way i see it in transmission microscopy, the desmosome has a set of proteins perpendicular to (and intersecting) the plasmalemma (desmocollins and desmogleins), then a row of proteins which act like lock washers or lock washers (plakoglobin, plakophilin and others I don’t even know about) that are pretty much perpendicular to the desmocollins and the desmogleins, and then another set of proteins, represented by desmoplakin, that becomes a row perpendicular to the to those “armadillo” proteins, and finally, the connection of desmoplakin with the intermediate (or other) filaments again perpendicular.  So there is a layering of different proteins, designed to accept all kinds of stress, and pulling, and shifting.

I have yet to find a diagram, out of literally thousands, which conveys this idea.

What’s more…. the fixation on the desmocollin desmoglein partnership creating the central dense line of the desmosome as being like a W does not fit the transmission microscopy either.  It would more likely be an S, again, rarely mentioned, except one pretty close approximation to what is seen (also an S and an Lambda shape).

A really disturbing depiction is the continued showing of intermediate filaments coming into the desmoplakin with a hairpin turn and back out again….. that just is not seen in TEM.

top diagram is what i have readily found in the literature…. mostly parallel depiction of the proteins which comprising the desmosome.  (black and colored vertical stripes) — which I think misses the point of the layering.  Most left hand bars are what I would predict, everything from the adjacent diagram to the right imply parallel arrangement of the proteins, including the intermediate filaments.

So this to me is more structurally sound, and we all know nature is pretty good at making changes to improve “soundness” LOL. So I would opt for this interepretation below. an explanation: plasmalemma (three layers blue and white); central band within the plasmalemma would be what is seen with TEM, i.e. the dense central line (dotted here) made by the association of desmocollins and desmogleins from each adjacent cell and this happens in the extracellular space; the armadillo repeat proteins, as many diagrams put (but not this one) are aligned more or less vertically along with the intracellular domains of desmocollins and desmogleins, but here I have emphasized the “lock washer, or washer” function holding two parts of this cellular rivet in place.  THen the desmoplakin molecules which in turn align perpendicularly to the area of the outer dense plaque proteins…. and finally again perpendicular to the desmoplakin… the intermediate filaments (green =  direction of orientation.

Breast cancer and the environment newsletter 6

Breast cancer and the Environment newsletters

Many xenoestrogens (chemicals which the body is tricked into thinking is estrogen) can pose risk for breast cancer. One such chemical is bisphenol- A, (Figure 1) which can interfere with normal estrogen-dependent functions. The increasing prevalence of xenoestrogens in the environment may partly explain the increasing incidence of breast cancer, though direct evidence is not overwhelming.

The use of bisphenol-A (BPA) in the production of plastics began around 1891 (e.g. in baby bottles, food containers, water main pipes, and laboratory and hospital equipment). Its estrogen-like (in this case, estrogen-disrupting) effects began to be noticed in the 1930s. Prenatal exposure to BPA (in rats and mice) changed mammary tissue and led to cancers in adulthood. Other animal studies used BPA to show that when breast cancer has been induced by a carcinogen that risk was increased further. If the animal studies correlate with humans, then even a small exposure to BPA could cause an increased risk for breast cancer.

Exposing cells in culture to BPA can cause neoplastic transformation of human breast epithelial cells.

“Consumer groups recommend that people wishing to lower their exposure to bisphenol A avoid canned food and polycarbonate plastic containers (which are identified as , unless the packaging indicates the plastic is bisphenol A-free. The National Toxicology Panel recommends avoiding microwaving food in plastic containers, putting plastics in the dishwasher, or using harsh detergents on plastics, to avoid leaching”. (thanks wiki)

Some estimate that 92% of canned goods with plastic liners have BPA. WHEN THE BRAND NAME on your canned soup is “Healthy Choice” and the BPA is 323 ppm, it’s kind of sad. I think its going to be a while before companies get the message…. therefore BUY FRESH
http://organicgrace.com
http://treehugger.com

HERE IS THE PDFPRG-DEH_Issue_6

Mechanotransducer (the desmosome)

Ultimate order, the cell

“Mechanotransduction is known to be an important biological process rendering tissues both resilient and responsive towards their mechanical environment” says the first line of the introduction of this article.”–   mechanotransuction: definition:(biology) Any cell, etc. that generates a measurable response to mechanical stimulation–so this definition includes many many organelles, including the desmosome.

It is fun to think about the desmosomal – mitochondrial tether as the energy supply and the

Adherens junctions and desmosomes

Desmosomal mitochondrial associations, The cell: images, Ultimate order, the cell

Narrowing of the intercellular space = about 5nm where the cadherins couple

I am not sure why the intercellular space is recorded by What When How as different dimensions (beside the adherens junctions 25nm vs 20nm, the height of the adherens junction itself) and of the intercellular space beside the cardiac desmosome as 35nm and the height of the desmosomal intercellular space as 20-25nm. It seems to me that if one is comparing intercellular space heights, that one really needs to set some parameters and get some comparisons. This could be a massive job, as there are so many variables, not to mention fixation parameters, tangential sections, membrane proteins, cell types and where on the plasmalemma one is going to attempt to measure.
Just in four desmosomes (between hepatocytes from syrian hamster) all fixed the same, similar ages and reasonably good section orientation the following is clear.
1. the intercellular space is pretty variable
2. desmosomes are going to have a slightly smaller intercellular dimension than adjacent intercellular space
3. the center dense line extends beyond the outer desomosomal plaque proteins
4. there is a density in the plasmalemmae as an annulus or ring around the desmosome but intercellular space is wide
5. the separation of leaflets of the desomosomal plasmalemma is just a little more distinct than distant plasmalemma
6. the outer leaflet of the plasmalemma at the desomosoe seems to be quite rigid

See below, four examples and the relative wide range of reduction in intercellular space. Syrian hamster — routine electron microscopy, red dots intercellular space remote from the desomosome, blue dots height of intercellular space at the desmosome. Two of the images – right top and bottom – have double mitochondrial tethers. Left side images have a single mitochondrial tether. Right top and bottom have cross and longitudinal sections of intermediate filaments, respectively adjacent to the mitochondria. In both cases the outer mitochondrial membrane also has a rigid look.

20,000 data points and no significant results

Rants

“20,000 data points and no significant results” this was a line from an episode of Big Bang Theory  (thank you screen writers.. ha ha).  Just so true as to be painful.

20,000 electron micrographs and no signifiant results could be the story (though i am exaggerating a little) of my career as a microscopist.  But truth be told, there are fair number of negatives that are over exposed, out of focus, scratched and water stained. Not to speak of those that have coarse staining with lead and uranium and are sectioned too thick or too thin to be totally useful.  And then there was the antique microscope I used (an old Siemens 1A) which required 14 manual movements (not including learning how to be ones own built-in human light meter) to usher through one acetate negative.  What fun.

I don’t know now (after 10 years) whether I can remember the sequence of motions to expose one picture, but it went something like this, record the magnification and block (grid) number, center the beam, then make sure the light is diffused evenly over the circular area marked on the screen and at the best contrast (the human light meter part), lower the little tiny central fluorescent circle piece that was the place to look at things a little more closely (with out changing the mag), flip the switch to block the beam, then lift the fluorescent screen (plate), use the knob on the lower right of the camera (large knob) to crank the plate under the beam, listen for the drop of the plate (with the film) into position  (btw, sometimes plates got jammed, this required closing off the camera (if the jammed casset would allow) letting air into the camera — in the dark, manually reaching into the camera box, touching up on the bottom part of the previous “stuck” film cassette till it dropped into the bottom of the camera and repumping the camera),  pressing the exposure button, cranking the handle on the camera backwards causing the plate to drop, lowering the fluorescent screen….  haha… that was the routine.  I might add, that it didnt take much more time than the digital picture taking, as it requires using the mouse to click, refresh, update, finding the folder for saving, waiting, and then going back to live screen (in fact the “drag” on the screen is horrible, compared to the smooth motion of looking live into the microscope)….  my feeling is that the film actually still had better imaging properties.   (yes, i still have more than 20,000 negatives). Would that i had known to take more time in the photographic part of the process always using the finest grain developer, being more precise in exposures and developing times, but i often resorted to dumb-ol-decktol  ha ha..and being in a hurry.  Decktol because it was cheap, being in a hurry because someone was beating the door down for results. That of course was short sighted.

Original negatives were glass plates, then with little inserts we could use acetate. THe scope could not be fitted with an automatic device for photographing…. otherwise i might have 100,000 negatives and no significant results (hahahah).

I googled for a picture of the Siemens Elmiskop 1A and up popped this reference, and this is MY OWN microscope, archived at UC, which I thought had been trashed and recycled when the building I worked in went down…..  Dontcha love how the users of this scope don’t get credit….. named here…. Nikolwitz, Bell, and then me,  Miller…. three users in a short 55 years. This photo is from room 310 of the original Kettering Lab that is now a parking lot.

This is a photo of my empty lab just before the building was demolished and the scope moved to a display in the Chemistry – Biology Library, along with the radiation tape I put on the scope myself (hahaha) and likely put the post it note on to cover up the “light” which interfered with looking down the finding scope.

Dr. Nikolwitz ( I havn’t spelled his name right) was so possessive of this scope that no one, that is NO ONE could touch it. Later came Dr. Mary Bell.  When she left, I became the individual who put it to use.  I really treasured the hours looking into that scope at the phenomenon that is “life” in all its uncanny, unparalleled, beautiful order.

You cannot imagine how back in the 1980s how difficult it was to get radiation safety to come and monitor the potential radiation leak from that scope.  I know that i worked on it actually pregnant, and kept bugging them to check it so i didn’t have to worry or cause damage to an unborn baby.  It was an old scope even at that time.

Sadly, there is a pitiful review of that microscope in the link below from my own institution, which says precious little about the contributions to science made on this instrument or respectfully names the PIs of the NIEHS Health Sciences Grant back in the late 1960s that made it possible (as a pdf). My guess is that the two instruction manuals which were referenced in the pdf posted at the URL below, were the two blue paper binders that sat on the brown shelves behind the scope (picture on the right).

There were hazards to using that scope, the diffusion pump had mercury in the oil.  The filament (which needed to be changed frequently, was at the top rounded part of the scope, and there was this very hefty static discharge arm, that I was always very very careful to have in place before I opened the top.

Get a grip on the size of that electrical cable, to cary 100,000 volts, and one time it gave way… that was the most expensive electrical cable I had every heard of…. nearlly 8000 back in the 1980s.

There was a dark room in the same room as the microscope, with safe lights for photograhy, and a developing sink and aeration baths for the glass and/or acetate film which had to be taken one by one out of a camera casette (just beneath the beam under the table part) that only held 12 sheets.  So this required a continual opening and closing of the column and since there were about as many film holders as casettes, it took a long time to take images.

No automatic printing of magnifcation bar — all this needed to be recorded by hand in the dark.  Besides that, film dropped from top down, so the last picture on the top was the first to be developed…. a constant issue…I took to putting a little rectangular box beside each change in grids and briefly sketching out the image that began a new set.

Loading the film was another part of the process…. and careful attention had to be paid to the emulsion side (in the dark again ( or with a small safelight)), one learned to note the “slightly sticky” feel to the emulsion side of the film and run ones fingers around the edge of the acetate (no such thing possible for glass) sheet for the notch…. I cant remember if it was upper left hand corner… but i think that is where it was.

Another hassle was the “pole piece” which dialed in different lenses thereby changing the magnification on the plate below. That, was important to record along with the other hand written metadata.

Aligning the scope was a big pain in the neck.  Every time a new filament was installed it required alignment, pulling out this diaphragm, using the two little turn screws to recenter, finding the “hotest” “brightest” spot before turning on the different lenses.  Then too, as the filament aged, the bright spot (at least in my hands) would tend to move, and realignment was necessary again.  Alignment was critical for focus, haha, no such thing as a “wobbler” on this scope.

There was the recirculator issues too.  Haha.  Big and bulky, aerosolizing some kind of freon (green) into the air while I worked.  Well…  it is past now. And for the short pdf written by Mr. William B. Jensen (from two google searches) that his name should appear so prominently by this old master microscope is just silly.

 


Seimens 1A transmission electron microscope: Elmiskop Electron Microscope

Easter bunny mitochondria

Mitochondria: electron microscopy, Science and art cover illustrations, The cell: images, Ultimate order, the cell

Please forgive, LOL, as this little mitochondrion which is from guinea pig liver, just was staring out from the page. You might notice that there are some (blue) regular protein molecules near the bottom right and left.  These are likely to be ATPsynthase all aligned in perfect order — see previous post with similar structures from guinea pig hepatocyte mitochondria.

Bridge or intermediate filaments?

Desmosomal mitochondrial associations, Mitochondria: electron microscopy, The cell: images, Ultimate order, the cell

When I first looked at this micrograph i thought perhaps the electron density under the mitochondrion (here above two desmosomes, tangentially cut and off center, was a bundle of intermediate filaments. That would be interesting since i don’t know if filament bundles likely composed of keratins 5 and 14, could make this tethering bridge between desmoplakin –part of the desmosome in complex with two remote points on the mitochonrdion.

Turns out that It looks more like a little invagination along part of the plasmalemma of the mitcohondrion, probably representing outer mitochondrial membrane. Red dit=ribosome@27nm, blue curley bracket=bridge, red lines, intermediate filaments bottom red lines, underscoring position of thedesmosomes.

electron microscopy mitochondrion

Outer mitochondrial membrane tethered to intermediate filaments

Desmosomal mitochondrial associations, The cell: images, Ultimate order, the cell

There is a reasonably prominent increase in density (and rigidity) of the inner and outer mitochondrial membranes where they are tethered to intermediate filaments tethered again with desmoplakin of the desmosome complex.  It occurs for the stretch which runs parallel to the desmosome and removed from it by about 140nm in height (as measured on these two micrographs – not ideal cross sections of desmosomes, so approximate dimensions).

Someone out there researching mitochondria membrane proteins probably knows which proteins these would be.  Top electron micrograph shows two separate mitochondria (tethered  — albeit tangentially) to the desomosomal complex showing intermediate filaments parallel to the plane of the plasmalemma in both instances. Red dot, ribosome=@27nm, red lines=intermediate filaments, blue curley brackets=inner mitochondrial membrane with increased density, green curley brackets=outer mitochondrial membrane with increased density at points of tethering. Dotted line in photo bottom left is where the outer mitochondrial membrane likely travels.

 

Desmosomal mitochondrial tethers

Desmosomal mitochondrial associations, The cell: images, Ultimate order, the cell

It makes a lot of sense that there are mirror images between two cells  — that is in terms of desmosomal / mitochondrial tethering. I have seen as many as six desmosomes tethered to two mitochondria (in juxtaposed cells). It seems that if the mitochondria supply the energy and the Ca+ regulation (for unzippering the desmocollin and desmoglein) and signals for dissolution and/or construction of desomsomes that one mitochondrion could do the job for three or more desmosomes.  I can envision the mitochondria alligned up along the lateral plasmalemma like soldiers standing at attention ready to build or destroy…

    1. seems pretty certain that transmembrane  portions of desmocollin and desmogleins affect the trilaminar architecture of the lateral plasmalemma, clearly seen on electron micrographs — they stiffen it….and widen it.
    2. There is an also an effect on the lateral plasmalemma in the annulus of the desmosome, appearing to be slightly more dense than plasmalemma further away from the desmosome, and also with slightly wider extracellular space (than non adhering areas) from outer lamina of the plasmalemma of each adjacent cells.
    3. Seems pretty likely too that most of the diagrams i have found in publications on the topic which bring intermediate filaments to the desmoplakin molecules at a parallel or nearly parallel position are just not accurate. I see lots and lots of perpendicular structures, that is desmoplakin perpendicular to IFs, rarely if ever one that looks like the desmoplakin and IFs are running the same direction. In fact, it doesn’t make sense architectural sense (in terms of adding resistance to pull and shear and separation to have them run the same direction. The long polymers (the IF) run parallel to the mitochondrion which really remains a fixed (seemingly) distance from the outer desmosomal plaque proteins…and plasmalemma. It looks likely that there are 4-6 IFs or more that run parallel to the plasmalemma (and outer membrane of the mitochondria)  not perpendicularly to it.  Just my thoughts (and what I see).
    4. Here is an electron micrograph which has cute little cross sections of IFs in the space between the mitochondria and the desmoplakin and outer desmosomal plaque proteins. (see the blue dots in micrograph on right, which I interpret to be IFs). Left hand image unretouched from scanned photo. (I could have added at least 5 more blue dots to the image on the right where blue dots are overlying cross sections of IFs.)

The new anti-christ: anti-social media

Apologetics, Rants

I used to think that insurance (yep, car insurance, health insurance, home owners insurance, social network) was the anti-christ. The reason I thought that was because insurance absolves us from culpability in our own mishaps, tragedies, and things that nature sends to us by allowing us to shift responsibility for things we can and sometimes cannot control and accept compensation from a nameless corporation(not always equitable compensation either, because cheaters do prosper). I feel we needed to be caring, and helpful, and understanding, and giving and bring forth from our own bounty to help others in need.

I think now that the anti-christ is social media because it perpetuates and multiplies unbridled falsehoods, opinions of a few, or many, that are radically antisocial and convey hatred, violence, brutality, unrealistic circumstances, and falsehoods, for ill gain.

Putting actions to my words, i have disabled my facebook, don’t subscribe to a newspaper, and believe what i see on TV is mostly hocus pocus.  I am not a proponent of conspiracy theories, i just think people are pretty dumb and i cannot be a part of 15million views of nonsense and potentially untrue and destructive garbage. nufsaid.