Category Archives: The cell: images

Isolated mitochondrion with smooth ER

A single isolated mitochondrion (blue)  and attached SER (pink) from a mouse at D28, likely isolated from liver,  postnatally having received NAC. This and other images were of controls for D28 mitochondrial pellets from hepatocyte specific GCLC ko mice also receiving NAC.  There is a lot of substructure going on at the attachment between the outer mitochondrial membrane and the SER membrane, and some tiny cristae right at that junctional tether. It is possible that a mitochondrial ribosome is in the field as well (dark dot rounded object in mitochodnrial matrix – middle right. (also there is a tiny piece of dirt, so dont be confused). So the periodicity between SER and mitochondrion seems to come out at about 35nm which is larger than I had hoped, but there is always the possibility that the structures were cut tangentially.

More on Form follows function in mitochondrial cristae organization

Not much text with this post, but here is a highly simplified, not real detailed, summary of what some think about mitochondrial cristae morphology. This is very basic, i intend to make some really nice diagrams including the inner mitochondrial membrane proteins as represented in 3D by the protein databases. That will include the OSPHOS proteins, and the channel and transport proteins and the cristae junction proteins.  But for now…. and blatantly missing several types of cristae (triangular in particular) here is a line diagram of cristae and some associated properties. The electron micrographs on the “aerobic” side of the diagram are from various images of hepatocyte mitochondria just cut out of existing micrographs, on the anaerobic side, the mitochondria are selected from isolated mitochondrial fractions. Both are traditionally prepared TEM processes. Abbrev: IMS, inter-membrane space; white rim area. Outer and inner mitochondrial membranes=black lines. Grey interior= mitochondrial matrix. Micrographs, not all the same scale,


While i love the tomographic 3D reconstructions of portions of the cristae, it seems that this is sort ofwhat most of us rendered in our minds before those data confirmed the structures.

 

Mitochondrial cristae junctions and pores?

Searching through micrographs to determine whether or not i can visualize a mitochondrial pore or crista junction.  Here is a cutout of a border of a mitochondrion which looked to have several adjacent and more or less evenly spaced cristae along one outer mitochondrial membrane. None of these exposes a clear connection where an inner mitochondrial membrane space should appear. Also of note (in this experimental set of animals) there tends to be a narrowing at the base of the cristae, and a little ballooning of the remainder of the cristae bring. This might be a manifestation of the cristae junction. In a stretch of outer mitochondrial membrane about 475nm in distance there were 6 areas where the former and the cristae membranes meet.

In this particular mouse liver electron micrograph the animal was a hepatocyte specific ko for GCLC, specifically negative 18408 block 78375 wc/ii animal#5 and 50 days old. red dot=@27nm (one cytoplasmic ribosome) used as a micrometer.

mitochondria liver electron micrograph cristae junction
Looking at the matrix of this mitochondrion several densities appear which may be too small of mitoribosomes.


Another mitochondrion which i examined and pseudocolored the same those above, has about 1 cristae membrane junction pore per 250nm or so counted over a stretch about five times that long. Clearly, physical state (these KO mice) nutrition, gender, cell type and function which the mitochondrion is supporting.  Green is the inter cristae membrane space,  pink is the outer and inner mitochondrial membranes, blu background is the mitochondrial matrix. The tissue from which these sections were made was a ko, (conditional KO of GCLC in liver).

Isolated mitochondrion

Just looking for the myriad features that should show up in mitochondrial ultrastructure. THis isolated mitochondrion is from a wild type mouse post natal day 14, liver.  It does show areas where cristae membranes (inner mitochondrial membrane) and outer mitochondrial membranes create the inner mitochondrial membrane space (green) and the membranes themselves (pink) and the matrix (blue).  Within the texture of the matrix it is tempting to try to find circular mDNA and mitoribosomes and there are hints of them here but nothing really obvious shows up. Trying to find some difference in inner mitochondrial membrane density which would indicate the crista junction is pretty disappointing as well.  Not all structures named, or presumed to be real, can be found in routine micrographs. There is one little circular structure in the middle of the matrix which I might well have included as cristae membrane. isolated mitochondrion cristae inner membrane

Crista junctions in mitochondria

I am trying to visualize crista junctions in mitochondria in the liver (mammalian). These might be visible here, in a KO mouse which has increased oxidative stress (previous posts). It looks to me like there is a small consistent area at the place around cristae approaching the outer mitochondrial membrane that looks “different”?  Find the 100nm markers (vertical at the edge of a crista and horizontal by the red dot over on the right hand side) are an approximate marker for the size of a cytoplasmic ribosome (27nm) and calculation for 100nm from that.

Cristae in this particular mitochondrion and many others show an increase in the amount of matrix space and a more vesicular type of cristae, and some times have cristae inclusions.

Mitochondrial ultrastructure

It is a little difficult to get the whole picture of mitochondrial ultrastructure together in one place and in one image. The the large and complex groups of proteins like ATP synthase,  or mDNA and mitoribosomes and the proteins for energy conversion, or calcium storage, ion transport, proteins that are involved in cell growth, division and apoptosis, as well as the basic mitochondrial shape and volume density of and shape of cristae and the amounts of mitochondrial matrix, and many other things of which I am sure i am not aware….are difficult to sort out.  Ultrastructural aspects related to these functions are not well understood, and at best poorly diagrammed.  I have found one website (biology by the numbers) which does do a great job of labeling size and giving measurements of some aspects of mitochondria, but of course not all, and not neatly organized. There really probably is not going to be much consensus about these variations in mitochondrial ultrastructure because the influence of tissue, cell, metabolic state, types of fixation, the embedment, as well as many variations in scope mag, deliberate manipulation of the images, poorly kept records, presumptions, and the list is endless.  So the question is how does one go about diagramming (or imaging) the most educational presentation about the mitochondrion. A really perplexing structure is the “pore”  or cristae junctions.

Add to that list of  variables, the long list of diseases where mitochondrial shape is found and the concept of imaging the “perfect educational” mitochondrion becomes more difficult.  So trying to find a great diagram came about because of an inclusion that I found in the cristae of some of my own micrographs.

It would just be nice to see these structures ( 5-10nm diameter) in the literature with the normal ultrastructure of the mitochondrion, which is not really that well documented for standard TEM images though there are some nice molecular diagrams of membrane proteins.

The round electron dense inclusions are unknown (in a crista space)(in these not-perfect micrographs). It is possible that in the bends of a couple of cristae, there are densities right at the bends which would seem to be identifiable.  Looking at the blue dots in the top image below, right at the curvature of a crista–one might see those dots as ATP synthase (blue dots are given at the approximate size that ATP synthase should be ( “biologybythenumbers” diagram (thank you for that) (bottom image).  Also, separate, larger orange dots could be mitoribosomes which reportedly are just smaller than cytoplasmic ribosomes (picture as red dots (taken from the same micrograph as seen on the left). A second reason for working on mitochondrial ultrastructure is to try to figure out what the electron dense (and homogeneous round) protein is within this crista space)

Metal? cristae inclusions in mitochondria

I found a paper that showed a TEM of a dense round intracrista inclusion that had some similarities to what is seen in the Gclcwc/ii hepatocyte specific KO mouse at 50 post natal days.  Paper is Characterization of Intracellular Inclusions in the Urothelium of Mice Exposed to Inorganic Arsenic Toxicological Sciences 137(1), 2013 by Puttappa Dodmane et al. I cut and pasted part of one of their electron micrographs of a mitochondrion with such an intracristae inclusion (right side of picture) next to what is seen in the KO mouse (my picture, left side of image).

 

Mitochondrial and ER inclusions, and nuclear invaginations in Gclc hepatocyte specific KO mice

Hmm. Is there a possibility that the inclusions in the inter-cristae space of mitochondria, the inclusions sometimes seen in nuclei (probably invaginations), and the iron-like dense inclusions in the ER hepatocyte cytoplasm, are linked by enzymes that are involved in the maturation of cellular Fe/S proteins for which mitochondria are important. These images are from Gclc hepatocyte specific KO mice, one image (with the nuclear invagination with iron-like spicules upper left) came from a KO that was rescued with NAC the other two micrographs are from wc/ii mice, unrescued, at day 50.

Puzzling? structures with structures

Aside from the fact that the word “structure” has been usurped by the molecular biologists from the microscopists, both show structure. The new puzzle is to fit the two together. I personally know nothing about protein structure but know a little bit about cell structure… so the deal is to find where the two can become one.  This of course is now accomplished by computational microscopy and better resolution, none of the equipment or funds for me to play with, BUT i still enjoy finding what is available online and working to understand that about which I am curious.

Beginning with the paracrystalline proteins in some mitochondrial disease states it became a puzzle to try to solve: that is, what proteins are making this brick-like inclusion in the matrix of mitochondria (see previous posts).

Just beginning with — Complex I (NADH:ubiquinone oxidoreductase) critical to energy metabolism in mammalian mitochondria on the inner mitochondrial membrane I used RCSB PDB to play with the purely “visual” issues of the protein. First diagram below…. shows a band in the transmembrane portion which by my eye is quite different than the rest of the structure in terms of color – and therefore leans toward hydrophobicity (according to the coloration charts of RCSB PDB). This model (which is to be taken “generically” of the NADH ubiquinone oxidoreductase I have shown on as a view from the nominal R and L (the bumby lump (arrow with no text) which I am assuming from the pale color is neither hydrophobic nor hydrophilic? just above the intramembrane “box” on the L). I doubt there is a “right or left” but for descriptive purposes, the front back – right left has been shown because they are so different, except for the transmembrane portion which is clearly a band that goes through and through. The inner mitochondrial membrane is from a diagram I did earlier… but gives a suggestion of the orientation of the NADH ubiquinone oxidoreductase that i have found for this in other diagrams online.

So now to add some other diagrams which give more fun images. This one, oriented identically to that one above, shows alpha helix area (bright pink–also according to the charts of the RCSB PDB viewer guide) that is within the bounds of the inner mitochondrial membrane. When one rotates the ribbon molecule, they are all neatly aligned perpendicular to the length of the membrane. I have to assume that they fit within the lipid realm of the trilaminar membrane.

Here is an edit to fit the group and an electron micrograph. Ha ha… probably too big…I will have to google the size of the molecule relative to the 3-5 nm thickness of the trilaminar membrane. within the band of the mitochondrial membrane it is easy to see a change in aminoacids by the increase in grey coloring in that area. This model is by element, the amino acids in the central region are mostly isoleucine, leucine, proline, and phenylalanine which RCSB has colored with very close shades of grey, hence the slighly more grey look to the transmembrane part of this molecule.

And the ribbon molecule emphasizes the vertical order in the transmembrane band….  I know there isn’t really anyone out there who doesn’t already know this, but the visualization here really makes it so apparent. Sheep – entire respiratory chain complex 1 ribbon diagram from a different database. .
and Cryo-EM structure of human respiratory supercomplex I1III2IV1