Monthly Archives: January 2018

Desmosomal mitochondrial tethers: fetal follicular cells of the thyroid

Just in case anyone is interested in whether there are desmosomes which are tethered to mitochondria via intermediate filaments in cells other than those I have reported so far then look at a fetal follicular cell (of the thyroid) and it is very apparent that these pretty large desmosomes with mitochondrial attachment via filaments is certainly happening. This site is by the tight junction, adherens junction, and the desmosome.  Just like hepatocyte D-M tethers, which tend to occur around bile canaliculi in hepatocytes.

Red M, mitochondrion; three arrows point to three different types of intercellular junctional complexes (including the desmosome and the mitochondrion (most left of the three); colloid in the lumen (light green shading) can be seen on the right hand side of the electron micrograph. Red dot is @ 27nm in diameter representing the size of a ribosome.

Does every protein precipitate down to a 10 nm glob with TEM?

When examining protein structure with TEM it doesn’t escape me how the patterns sometimes fit into these very regular but seemingly not relevant patterns.  In looking at the densities which appear in the center portion of the desmosome and also the diameter of cross sectional cuts of the intermediate filaments associated with desmosomes, i get confused as to why both look to be about 10nm.

I have tried to google such phrases as “texture of lead citrate staining” with TEM, or uranyl acetate or osmium textoxide, or some other texture analysis that might put a “cap” on the limits of where one can go when measuring protein patterns with TEM.

Any images that I review for protein patterns I am forced to find adjacent areas and see if similar patterns occur.

It makes sense that fixation conditions will greatly influence the final texture of the tissue, and I doubt whether there have been any well controlled morphometric studies comparing the myriad methods for processing tissues for TEM. I found an article bu R. W. Horn on this and here is the first two lines as as quote “For the practical biologist applying electron microscopy to the study of biological macromolecules, there are serious problems in obtaining high resolution images showing detail below 2.5–3.0nm. The limitation in resolution from biological specimens can be attributed to support film thickness and granularity, specimen preparation, irradiation damage, focusing effects and possible contamination in the electron beam.” which i will not read. (Somehow it seems a travesty that Wiley (Bruker) think that I should pay to read a pdf the data for which were mostly like funded with public funds — i wont pay). But some terminology and also the recognition of 3nm being an important “stop” point beyond which any assessment of rhythm or pattern in a micrograph would just be “gestimate” is reasonable. I actually, at least for my micrographs, probably from negatives not developed with the most fine grain developer (a penny wise and pound foolish choice made in my career to not use the most fine grain developers for my negatives and prints…I was at that time unable to think to the future of microscopy and structural protein biology and the links possible between the two fields, so sad really).
So here is a micrograph of a mouse hepatocyte desmosome which is cut tangentially, the central dense line where the desmocollin and desmoglein come together (but it is still slightly visible), but there areas where the latter two molecules traverse the plasmalemma, there is a little density… at least I think it is periodic. I have added black arrows to indicate where i see those periodicities in the outer lamina of the trilaminar membrane, and moved that same set of arrows to different sites in the micrograph (in this case both are intra-mitochodrial) to see if by chance the same periodicity might arise (thus helping to convince myself that the periodicity actually means something). You can see that the red and blue arrows dont line up exactly with any matrix proteins in the mitochondria.

More desmosomal mitochondrial tether samples

Mostly, if not all, hepatocytes from a variety of species. Some of these images have the species designation in a lower case letter as follows: r=rhesus, st=stub-tail, m=mouse, oops, i might have put r for rat somewhere, f=ferret, gp=guinea pig.  May not all be present but must a few are marked.  I am looking for species differences but these are not ideal images for such detail and are just archived samples collected in 40 years of microscopy.

There are certainly differences in size, and also number of mitochondrial tethers that can be seen, and the biggest nicest desmosomes I have seen are in tongue (image below is bovine i believe — thank you to whomever’s image that is), and the most numerous of course in skin. I havn’t bothered to add micron markers…. it seems to be quite variable depending upon where in the “disc, or spot” the section occurs.  The mitochondria and cristi give relevance to magnification anyway.

 

This made me laugh so hard

This made me laugh so hard i boosted my immune system for a full week.

….Actually, throughout my life, my two greatest assets have been mental stability and being, like, really smart. Crooked Hillary Clinton also played these cards very hard and, as everyone knows, went down in flames. I went from VERY successful businessman, to top T.V. Star…..

I think overall the health of all americans will improve just because we laugh at your tweets.

Mitochondrial-intermediate filament functions… only one at a time? probably not

I found a nice review article the title is “Intermediate Filaments as Organizers of Cellular Space: How They Affect Mitochondrial Structure and Function by Nicole Schwarz * and Rudolf E. Leube”  free online, about intermediate filaments and mitochondria, which is part of my reference list as I am trying to figure out where there are species differences in the ultrastructure of desmosomal-intermediate filament-mitochondrial tethers.  So this is a nice diagram but i think it is too primitive, or perhaps too limited, or maybe just has not been taken to the next step.

This image is their Figure 1 which shows three possible intermediate filament – mitochondrial arrangements.  My exception to it is the following : there are areas around each mitochondria where all these types of bonding-binding-exchange take place, at least it seems to me.  For instance, the binding of intermediate filaments to the outer mitochondrial membrane (OMM) probably doesn’t occur (at least in hepatocytes) in a complete manner surrounding the entirety of the outer mitochondria membrane, but rather definitely is “focal” as it looks when it is tethered to another cytoplasmic organelle such as the outer edges of the nuclear pore, or the inner plaque of the desmosome.  There is a definite change in the structure of the outer mitochondrial membrane, gross enough that it is seen with TEM, and looks like a thickening of the OMM and a “flattening” out of that area which is engaged with intermediate filaments. There is also a shape change to the mitochondria when they are bound (i prefer the word tethered) to the filaments.  (so still talking hepatocytes here). Intermediate filaments positioned remotely from the OMM and called “signaling” is something that I don’t believe would be seen physically.  As for confinement, there are certainly areas of confinement of organelles, and they might be called “no fly” zones, I also need to find an example where the mitochondria are almost repelled by intermediate filaments.

In a couple of mouse models i have looked at, intermediate filaments themselves can be repelled (or it looks that way), of which one is the central confinement of intermediate filaments in small intestine microvilli  where the gastric HKatpase is missing).  There are a dozen, at least, intermediate filament x OMM sites that can be seen hepatocytes where there is obvious “tethering”, and i bet signaling as well.  So these categories might have a lot of crossover.

I am thinking that in order to signal, the intermediate filaments need to be physically closer to the mitochondria than this diagram projects…. I wonder how that could be determined with TEM.

 

Desmosomal-mitochondrial tether Stub tail monkey hepatocyte

This particular electron micrograph of a desmosome with mitochondria tethered to either side shows some nice orientation and detail. Particularly the intercellular space has the zipper lines that are the desmocollins and desmogleins. These lines have some regularity, but owing to the enormous numbers of possible orientation that one could get in TEM, it is not that likely that a perfect one will ever arise. I even consider the roundness of the “spot” desmosme and the possibility that the organization is radial, wouldn’t that be fun. Someone out there with 3D imaging skills could certainly test this with the molecular models that do exist.  I think it would be just as fun as looking at the tomographs of thicker sections.  Brown dots are likely areas where desmocollins and desmogleins are intersecting-interacting, and these represent the intercellular central dense line of the desmosome. Black lines are areas where the 5-repeats in desmocollin and desmoglein (i suspect) are spanning the intercellular space. The black dots are some kind of periodicity visible on the outer lamina of the trilaminar plasmalemma.  I didn’t find any good cross sections of intermediate filaments up near the mitochondria…. though I though they were there as dense elongated areas, not nice round cross sections.  (BTW… love the two eyes — aka intramitochondrial granules… these actually are very likely arranged strategically within the mitochondria near places of tethering…  would love to know where and why). Red circles are around little interesting radial symmetries… that showed up…

Anyway, this micrograph and inset are from a Stub tail monkey, which was, for all intent and purposes, a control, thought it did receive a tiny test dose of artificial blood.


Human hepatocyte desmosome

I have no clue, nor any record of how and why I ended up with a couple of blocks of human liver (most notably taken while i worked at childrens hospital) no way to trace the origina at all.  It is kind of interesting, this sample had lots of glycogen, also very dense mitochondria (might have preserved in a different laboratory — actually definitely by someone else since I never did any tissue exams on human tissue). The desmosome (which is not quite a double-mitochondrial tether (only one clearly tethered but the mitochondria in the adjacent cell is obviously tethered just down (or up) in the same block. This desmosomal mitochodnria tether is, like many, just adjacent to the bile canaliculus.

Periodicity in mouse hepatocyte desmosome

I measured the periodicity that I saw (looking like a little zipper) on the outer plasmalemmal membranes of two adjacent hepatocytes at a desmosome (green dots). They seemed to be spaced about 19nm apart, and were small (maybe 7 or 8 nm densities).  In addition the intercellular space in this desmosme really had a nice alternating linear look where the desmocollins and desmogleins would lie. Micrograph on the left, unretouched mouse hepatocytes and desmosome (mitochondrial tethered on the upper left, but somewhat inconspicuous), green dots, zipper densities, red spots, ribosomes, lines, likely to be intermediate filaments.

Rhesus monkey hepatocyte desmosome

This particular micrograph is too tangential to the desmosome to show a lot of detail but it does show some cross sections of what I presume to be (because the size is about 10-11nm) intermediate filaments. These crossectional “dots” of IF are in the right position above the inner desmosomal plaque.  Red dots = approximate ribosomal size (27nm) , blue dots = approximate IT cross section (about 11nm).  Micron marker is 100nm for both images. Image on the left is unretouched, image on the right has dots over ribosomes and intermediate filaments for comparison with image on the left. Rhesus monkey, control biopsy, before the administration of perfluorochemical blood substitute, #71 female, fixative=modified Karnovsky’s (isoosmolar Chick-fix), Millonigs uffer, 2% osmium tetroxide, EPON 812.

Spirit

i do not know if i have ‘spirit’
i want spirit
if i did have spirit then my life would be complete
i am open — but also somehow also self-closed to spirit
i try to listen to spirit
i listen to the spirit of El Elyon
the unknowable
the incalculable
the infinite
the eternal
but if i ever understand that spirit
it will no longer have spirit-power
since what i comprehend can never be awesome