Category Archives: Nuclear symmetry during apoptosis

Nuclear symmetry during apoptosis in all kinds of cells

Little green aliens among us

14CoS ko, hepatocytes, rescue with NTBC, Electron micrographs of liver, Nuclear symmetry during apoptosis, Science and art cover illustrations, The cell: images, Ultimate order, the cell

So sorry, ha ha, but when i see this kind of nuclear symmetry (an electron micrograph, with two little presumptive eyeballs staring at me (condensed chromatin), zygomatic arches coming off (as condensed chromatin on the sides of the nucleus), and a hairline part at the center of the frontal bone (condensed chromatin and nuclear pores on the inner nuclear membrane at the top of the nucleus) and a pointy chin, I am compelled to laugh out loud and add this to my collection of TEM-Devils, and sometimes other objects like hearts and ghosts and snowflakes. Anyway, here is a little green cutie, which spawns from a mouse hepatocyte null for 14CoS and not receiving NTBC as a “saving” medicine and prone therefore to undergo massive hepatocytic apoptosis within a day or so of birth, and ultimate demise. This mouse is 24 hours post partum and showing signs of hepatocyte apoptosis, and mitochondrial and ER disarray, neg 16042, block 65718, anm#5, one chatter line removed with photoshop, layers constructed in photoshop, pseudocolor and eyes added in CorelDRAW.  Dont you wish there was a place to publish such stuff…. Maybe i will start my own journal…  help me think of a name.  (LOL)

 

Endstage apoptosis in hepatocytes

14CoS ko, hepatocytes, rescue with NTBC, Electron micrographs of liver, Nuclear symmetry during apoptosis, The cell: images

Here is a textureless (or nearly so) grouping of DNA around the periphery of an hepatocyte from a 14CoS ko hepatocyte, from a young mouse not rescued with NTBC.  Symmetry to clumps or peripheral digested DNA is obvious.  There remains some substructure to the “euchromatin” in the nucleus as well as some structure to the areas of presumptive digested chromatin.

Asymmetrical apoptosis with four blips

Nuclear pores and nuclear architecture, Nuclear symmetry during apoptosis, Nucleoar organization, Ultimate order, the cell

I ask forbearance for my posting this. I have seen this asymmetrical type of apoptosis in some experiments that were done while investigating an anti-apoptotic gene. I was just doing the electron microscopy, others were doing the cell culture and knockdown experiments.  Typically apoptosis in culture presents with a round nucleus lots of large nucleoli and a bilateral (mostly) distribution of granular material along the inner nuclear membrane of the cell as it dismantles itself. The squeezing and pinching in this type of apoptotic cell was just really unusual and as is always the case on government grants, the reasons were never flushed out because of time and money constraints.unusual apoptosis in knockdown tissue culture experiment A549 cells I bet there is a really interesting reason for these, and this particular set of culture dishes got a scrambled version of an si67 probe and was supposed to be a control. It seems to me that part of the nucleus a just got extruded? I did count 8 nuclear pores (main criterion being an obvious set of cytoplasmic filaments off the cytoplasmic side of the pore.  Few mitochondria, many vesicles. One interesting thing about this apoptotic style is that the cytoplasm is not full of ribosomes as occurs in apoptosis in many cell types.

It is not something i could have made up (LOL) but my initial guess is that the four little ball-antennae are some remains of cajal bodies, and heretically i could suggest that the linear divisions radiating out into the “skirt” of whats left of the nucleus, likely has something important to do with each chromosomal territory (separations, walls, scaffolding, call it what you like).

 

Interchromatin granule cluster ?

Nuclear pores and nuclear architecture, Nuclear symmetry during apoptosis, Nucleoar organization, The cell: images, Ultimate order, the cell

Nothing is black and white, except electron micrographs, and even then they are easy game for dodging, blending, blurring, mending, and pseudocoloring. Cytoplasm here is blue. In this case I have a cell from a culture of A549 cells in which C9orf82 was knocked down using si67.  This is pellet 4 from one of those studies. It produced this cell in which the granular component of the nucleolus was separated into at least two bodies (almost bilaterally symmetrically arranged) which rest at the inner nuclear membrane. Fibrillar centers are small, dense fibrillar component is still visible (albeit not distinctly) in the nucleolus (pseudocolored purple in the center of the cell).  Off to the side is what I could guess would be either degraded DNA (not my first choice as an answer (it is pseudocolored bile-yellow) or perhaps a fine granular component of the nucleolus in an apoptotic cell.  Part of an interchromatin granule (red, lower left) cluster (IGC) (sometimes called speckles, a name which is not really to my liking since the IGC has a boundary, a background texture that is also part of the structure).  So I would propose keeping two names, the “IGC” which is the greater boundary of that area and “nuclear speckles” as well, since the densities (as seen in this micrograph) can apparently be large.

If i am not off base here, the background of the whole IGC is red, but there are very clearly larger than typical speckles within the IGC, and the latter itself is quite small.  With immunohistochemistry the diffuse staining of IGC background proteins fluoresces with some of the antibodies to proteins like SC-37 in an area which can be 2 microns across easily.  I am looking for alternative proteins stained in very punctate regions of the IGC and hopefully they will be different and good markers for a separate ultrastructural components of the IGC. The point would be to localize to the densities with the IGC, some proteins for splicing, and accept that the diffuse staining could apply to SC-37, but not other proteins, mainly those in the variable size granules within the IGC.

We will see if there are data to back up the keeping of both names but assigning them to their obvious separate entities. BTW, there is pretty obvious bilateral symmetry to this apoptotic cell nucleus. and a radial symmetry to the dense bodies within the red colored IGC. And while i have no clue what the curley-Qs are… i bet they have something to do with the transfection. Just looking at this cell shows one tiny surviving mitochondrion, in this apoptotic cell, which is very close to the cytoplasmic filaments of a nuclear pore…also something which needs to be monitored during apoptosis.

Components of the interchromatin granule clusters are ??? and do they change during apoptosis

Nuclear symmetry during apoptosis, Nucleoar organization, Ultimate order, the cell

electron micrograph A549 cell knock down antiapoptotic geneWhile interchromatin granule clusters were thought to be the “the same structure” as nuclear speckles, i wonder if that opinion was justified at the time, I am not about to make that leap. I need to sort this out, since with plastic sections stained with toluidine blue, the interchromatin granules really were not “lucent or unstained” areas, but a light tan color, if i remember right, and with TEM they were actually more closely punctate than usual euchromatic nucleoplasm. So the connection needs to be further varified…and this might be why there are mixed notations and confusion and disparate explanations as to what the speckles and interchromatin granule morphologies identify. Also, what I have seen referred to as interchromatin granule clusters have two (at least) maybe three or four sizes of granular elements depending upon metabolic or functional state of the nucleus is (as in G,S and also stages of apoptosis, maybe necrosis as well.  (Of course all the (11 so far) listed processes of cell death and cellular-self destruction could be listed here but I just subscribe to the main ones, necrosis and apoptosis for now.) This cell has nucleolus dark blue, nucleus golden, cell cytoplasm greyscale,  interchromatin granule cluster of pink pink, and white box shows area enlarged in picture below. The larger (one quite large) and four or so smaller clusters can certainly point to some metabolic phenomenon which is not really occurring in a non-cultured-non-exposed cell.

The purpose here is to comment on the various sizes and shapes of such densities within the interchromatin granule clusters, and to examine transmission electron micrographs of so many cell-death projects to see whether concistent patterns interchromatin granule clusters have components change in size, position, density, and shape depending on which processes are occurring within the nucleus.

In particular, when A549 cells were examined after knockdown of an antiapoptotic gene (using si67), then the interchromatin granule clusters contained large aggregated densities (large in comparison to the smaller entities in untreated cells).  Here I have localized these in a single cell (transmission electron micrograph of the area bounded by the white box above), just to point out what is observed. Relevance is yet to be determined for these larger than usual areas of of conspicuous density within interchromatin granule clusters. The curved arrow points to bar-like organization of a filament or fibrilar area (sometimes this ribbon type linear organization is seen in cajal bodies) and the width of these bar-like fibrilar structures would be something around 30-40 nm, if compared to the size of a ribosome (red dot in the center of the text that says 500 nm).  The large structure (electron dense) beneath the interchromatin granule is the top part of the nucleolus seen in the image above. neg 18272 block 78932 A549 cell si67 knock down of antiapoptotic gene in vitro. electron micrograph A549 cell knock down antiapoptotic gene interchromatin granule cluster)

A549 cell in apoptosis

Nuclear symmetry during apoptosis, The cell: images, Ultimate order, the cell

A549 cell in apoptosis: RNAi knockdown of C9orf82 induced apoptosis in A-
549 cells. Huge granular center of the nucleolus with very little differentiation or dense fibrillar area is seen in middle right part of the micrograph (purple) and a part of the nucleolus with tiny light fibrillar centers and only a small amount of the dense fibrillar component. A portion of the nucleolus is off to the left near a nuclear pore. The cytoplasm of this cell has some RER that does have ribosomes, mostly near the nuclear membrane, and only three sort of not to clear mitochondria exist, one of which is apposed to an nuclear pore. There is very little condensed chromatin around the periphery of the nucleus, mostly euchromatin which is very coarse. Some perichronatin granules, and a little bit of interchromatin granule cluster (speckles) looking like a baseball cap above the circular structures of the nucleolus. Within, perhaps is something dense (don’t know whether to call this a paraspeckle or not (the location within the speckle fits this description). Nuclear chromatin and nucleolus purple, nucleus itself (which includes the interchromatin granule area) orange. 18272_78932_a549_4_si67_apoptosis

electron micrograph nucleus RNAi apoptosis

Fibril, filament, snRNPs, granular component etc

14CoS ko, hepatocytes, rescue with NTBC, Electron micrographs of liver, Nuclear pores and nuclear architecture, Nuclear symmetry during apoptosis, Nucleoar organization, The cell: images, Ultimate order, the cell

This micrograph I am using to determine the sizes of the different nuclear structures, so this is the first attempt to define at least four different sizes of components. For me, there is similarity in the size of the granules (beads on a string maybe something like 30 nm) in the cajal body and in the nucleolus. Arrows point to the kind of layered banding parallel strand look of the cajal bodies,  the red circle is about the size of a cytoplasmic ribosome (which would be something like 27nm) and the diameter of the rounded object beside the cajal body (something on the order of 60nm) and an even larger fibrillar component seen through out the fibrillar centers and also just a little above center left (orange spot) which might be 130 – 150nm.  So there are four measurements of fibrillar components in this nucleus, so far.  Will post more.

electron micrograph liver 14CoS ko nucleolar architecture

Nuclear component N symmetry

Nuclear symmetry during apoptosis, The cell: images, Ultimate order, the cell

I found the first ever reference I have read that indicates that there is nuclear and nuclear symmetry within the same cell.  This is something i have observed (i believe) and would love to draw attention to.  So here is an article from 1971, readily available online, by one of the oldies of electron microscopy and published back in the hay-day of TEM.  It clearly states that the studies of Phillips and Phillips (1969, in JCB) using cells in culture found a similarity between nucleoli of the same nucleus.

So here is a quick cut and paste from a publication in Micron which shows what I found in HeLa cells, to me (see left, original, and right, areas of bilateral symmetry with lines).  To me this is quite unmistakable.  So will work on this.   My thought is that it will ultimately relate to the N, or the ploidy of the cell.

Mono ribosomes

Nuclear pores and nuclear architecture, Nuclear symmetry during apoptosis, Nucleoar organization, The cell: images, Ultimate order, the cell

Cytoplasm of an HeLa cell grown in vitro, no UV exposure, but inhibitor of caspase 1 added.  Lots of monoribosomes in the cytoplasm.  So this is still part of a study to summarize the ultrastructure o the nucleus, nucleolus, in apoptosis. This nucleus has a very large nucleolus, large fibrillar centers, not that much dense fibrillar component but a large granular component. Not a great micrograph, but data, none-the-less.