Electron tomography of nematode and human tissue culture cell spindles
Creators: name: Fabig, Gunar
Publisher: name: Zenodo
Year: 2025

description: Thank you very much Gunnar for agreeing to give us a talk today about this. We're really looking forward to it. Electroentomography.

 

And as I know you've kindly humored me and put a lot of technical specifications in here. So thank you very much. The floor is yours.

 

Take it away. Okay, thanks. So hello everyone.

 

I'm Gunnar Fabisch. I'm a postdoc in Thomas Möller Reiter's lab and I walk you through our workflow of electroentomography, how we analyze spindles and finally how to get from electron tomograms to numbers. So first I put a little bit of biology in.

 

I hope it's not too much. But we do this all to answer some biological questions. So I think it's good to have the background.

 

And so introducing spindles and male meiosis and C. elegans. And of course then I will focus on the methods, a little bit of hyperpressure freezing, free substitution and then of course all this reconstruction and analysis which is key for our approach. So our lab focuses on cell division and chromosome segregation and spindles in general.

 

And we use different model systems in the lab which is nematodes, C. elegans mainly but also like another nematode which is called our nemarodensis. And I think it's just an emergent model system which has three sexes. So it's a weird worm which has a male, female and hermaphrodite and it has also some interesting spindle properties.

 

And then in addition we study spindles in human tissue culture cells, mainly HeLa. And now we started also in U2S and RPE1 cells. And finally we want to understand how chromosome segregation works and how spindles are organized on an altostructural level and also how it is interplaced with the dynamic of chromosome segregation.

 

Okay so just to quickly recap, a spindle and metaphase is looking like that in a textbook view. So we have two centrosomes which consists of two centrioles and then from this microtubules grow out and polymerize. And in the center of the cell there are the chromosomes and metaphase and this green thing here is a multi-protein complex at the chromosomes called the kinetochore where microtubules can attach and if they do so we term them kinetochore microtubules.

 

Those are the red ones. And there are different classes of microtubules and we usually term them spindle microtubules if they grow towards the spindle but not attached to the kinetochore. And also astromicrotubules which grow to the opposite side and make contract to the cell membrane and anchor the spindle in the cell.

 

And after metaphase is finished and chromosomes are nicely aligned then the spindle goes into anaphase and there are two distinct anaphases. So one is called an anaphase A and it's characterized by the shortening of the pole to chromosome distance and like in the canonical view this is thought to be driven by a shortening of the kinetochore microtubules. So chromosomes move towards the pole by shortening of this k-fibers as we call them.

 

And another anaphase is called anaphase B and this is characterized by an increase of the pole to pole distance. So both poles move towards the periphery and drag the chromosomes with them. So and usually chromosomes are moved by either anaphase A or B or a combination of both.

 

And as we studied chromosome movement in the male myotic spindle in C. elegans I just want to briefly introduce what's like special about the cell division. So worms or meiosis in general is characterized by one round of chromosome duplication followed by two divisions. So we end up with a haploid set of chromosomes.

 

And in meiosis one is in the special thing is that the homologous chromosomes pair and get segregated. And the special thing about worms is that male worms have only five autosomes and one X chromosome which is determining the sex. So if there's only one X chromosome present the worm will develop into a male and if there are two X chromosomes present in C. elegans it will develop into a morphoid.

 

And like in the cartoon view it looks like that. So first the homologs pair and arrange in the center then they get segregated to the pole and the single sex chromosome with X stays in the center. And then the centrosomes even split and only afterwards this X chromosome segregates.

 

And we investigated that by EM. And I will show you the results of this later. And then this is followed by the second round of meiosis which is in principle like a mitotic division.

 

So the sister chromatids align in the center of the cell and they segregate. And afterwards the spindle poles are like shut down and the four sperm cells pinch off a structure called the residual body where everything is dumped which is not needed for sperm function. That's just the background of the spindles I will show you.

 

Okay so first what we did was some light microscopy. So in principle we immobilized the worms and did for instance light microscopy. And that's how it looks like after some pre-processing and segmentation.

 

So what you see here is gamma tubulin the spindle poles and in green and the chromosomes in red. And what you can hopefully nicely appreciate is that like the chromosomes move apart and the X stays in the center and only later it like snaps to one side. And this is a random process as far as we could determine that.

 

So it's not really you cannot like say beforehand where it will go. May I ask a question? Sure. If you go back to the previous slide.

 

Yeah here you show that the X chromosome is in the first division it's going into one cell and the other cell has none. Does that mean finally you have from the four sperm you have one with X chromosome and three without? No no no it's also it's also splitting here. So you have in the in final in the final product two with the X chromosome or with the X chromatids and two without.

 

Okay thank you. That means like the sperm which is like which is not having the X chromosome will produce a male in after fertilization again. Okay.

 

And then we analyze the like spindle dynamics of this division. So in this in this case here we as the centrosomes are marked with a GFP we could easily measure the pole to pole distance of the two GFP signals and also the pole to autosome distance and the autosome to autosome distance. So you can like with this kind of analysis you can really get the insights in the chromosome movement and properties of spindle dynamics.

 

And I just like want to mention this quickly but what we found was that there is an NFase B so the pole to pole distance increases and there's also an NFase A that means the pole to autosome distance decreases after NFase onset. This was kind of the surprise to us because in C. elegans embryo it was thought there was only NFase B without NFase A but in the male meiodic system apparently there is NFase A. Okay and then we also quantified the volume of the centrosome so the centrosome is quickly shrinking upon NFase initiation so it's it seems like the centrosomes are actively degraded and split. Okay but now as we now have the light markers to be analyzed we also want to have some insights in the in the alpha structure of the spindles and for that we now turn to electron tomography.

 

So for that the first thing we usually do in this process is we do high pressure freezing and I mean I don't want to mention too much about it because I want to give a whole talk about it. So in principle we freeze the samples very fast under high pressure and this leads to vitrification of the samples so we it prevents the formation of hexagonal ice and ice damage. And for that our instrument is the Volvent HPF Compact 03 which we got like two years ago so we are we are very happy with this machine I like it very much and it produces very good results constantly.

 

And what we have to do next is we have to select the the worm of interest. So this is how the elegance looks like on a plate you have to pick the worms which you're interested in in some cases like hermaphrodites or in my case I was interested in the male meiosis so you have to select a male worm which can be distinguished by this male's hail which is absent in the hermaphrodite or like if you need some practice for that so it's maybe not so obvious in this image. Okay so what we do then for the cryo-immobilization is we use the sandwich method.

 

So in in this Volvent machine usually we use the sandwich of two aluminum carriers one has an 0.1 0.2 cavity and one is like a flat and an 0.3 and we usually dip them in hexadecene so this is also cryoprotectant but it makes it also very easy to open them in the under liquid nitrogen. And so what what we have to do first is we have to put the like freezing medium into the 0.1 cavity and as a freezing freezing medium we usually use 20 percent DSA in M9 or 10 percent PVP and so recently we switched to PVP if you want to have a clean sample after the freezing because the DSA tends to stick to the samples and sometimes this is helpful for example if we look for C. elegans embryos in this capillaries we don't want them to like fall out easily in the free substitution but for for other purposes if you want to have nicely clean worms for example or fix them the PVP is also very good because it's kind of dissolved in the free stuff and then we have the clean samples afterwards. Okay so then the nematodes have to be like picked into the in this into this drop so you can kind of see them here and then the sandwich is closed with this flat side of the of this left carrier here and then the sandwich is put to the high pressure freezing holder is then closed and then it's quickly frozen and the fingers have to be crossed that the freezing is going well but then like samples can be stored in the liquid nitrogen for until further processing.

 

Okay for cells it's a little bit different and so for freezing cells we usually grow them in this flask here and we synchronize them by hitting the flask on the table so this will like float off all this dead cells or rounded cells and leave only attached cells then we incubate or after washing we incubate the cells for a certain time allow them to go into mitosis and then do this shake off again and collect the cells and concentrate them by by centrifugation and then put them to an incubation chamber and where they settle on sapphire discs and the sapphire discs we treat with after cleaning with some polylysine which makes them sticky and also with fibronectin which we found out makes make the cells stick much better to it and for the treating actually we we use this rubber pad so if you ever work with sapphire discs I can really recommend that this is just a piece of table tennis rubber which we just cut into pieces and then the sapphire discs nicely adhere but it's not too sticky to get them off so it's actually a nice support for for coating. Okay and then what my colleague Robert Kiewicz developed is a 3D printed chamber where we can incubate the sapphire discs much much better and it's much more efficient. So this is how this chamber looks like so it's a small cavity which has space for four sapphire discs and we can place them here and then fill them with this very concentrated cell suspension and we allow them to settle for a certain time and then they go into into division and then we freeze them.

 

So in the sandwich we are using is a kind of a different from the double sapphire disc method so it this is a carrier from Volvent which has an 0.04 cavity at the top and the sapphire discs with the cells go like reversed on on top of that and this allows for checking if there are trapped air bubbles but it also gives a very good freezing results. So it's not really like it's not really possible to film the cells but you can visually inspect if there are trapped air bubbles. And so for that we usually like put the carrier in the holder already put some freezing medium in and this is in our case for the cells a DMIM with 10% BSA and 10% FPS and then the sapphire discs are grabbed with a tweezer quickly dipped in the freezing medium and then like assembled with the cells facing downwards on this thing and once the once the sapphire disc is on you can easily see if there's an air bubble or not and if if there is then there's no no point to freezing it and we usually discard it because the air will either like destroy the sapphire glass or lead to really bad freezing.

 

Okay then sample holder is closed it's frozen. I have a quick question so so is that the normal sample holder that you are using here or is it a special one? I think that's the standard holder for three millimeter assemblies so this is the same holder we also use for worms so it's not a special holder because because the sandwich assembly here is also I think one one millimeter thick so it fits for both. Okay after the samples are frozen and in this case the worms we go to our free substitution protocol so in normal protocol we use one percent osmium and 0.1 percent uranyl acetate and acetone and our free sub routine is as follows so we keep them for one hour at minus 90 then we ramp up to minus 30 with five degrees per hour and keep the sample at minus 30 for five hours and then ramp up to zero and afterwards and we wash with acetone of course to remove the osmium and infiltrate in steps with epon arosite that's our resin of choice because it's very easy and stable in the electron beam photomerography and so the worms are then flat embedded between two teflon coated glass slides so like kind of we have some space in here with parafilm and then we just put the two glass slides together and polymerize in the oven for 48 hours and this method has a big advantage because you can afterwards really screen for the samples you want to section and for example here is now a male worm nicely embedded in resin and you can like trim or select a region to trim later beforehand.

 

Okay so of course when you do flat embedding you have to mount your samples back to a dummy plug for sectioning so for that we use usually like two component glue which is hardening pretty quickly then the sample can be trimmed and then we usually section like 300-350 nanometer thick sections and collect them on this foam ball coated slot grid which we don't carbon coat. I know that some people do but we usually don't do that. Okay the cell protocol is in principle the same with a few differences so we just recently developed a protocol where we use some histological dyes in the acetone so what we what we do is similar to a protocol published by Miriam Lucas and Roger Weff I think some years ago in one of Thomas' book chapters and this is also what we do so we saturate the acetone with a dye put it to minus 80 and then we filter the solution and we use then this filtered acetone to put the uranyl acetate in and then the following steps are the same just we do the embedding in this capsules from Leica this flow through capsules where the sapphire glass can go to the bottom and then we fill up with the resin.

 

And of course then the sapphire glass has to be removed and we Robert developed a nice method for that so what we have to do first is we have to trim away the the resin at the edges of the sapphire glass and then we just quickly dip the tip of the sample with a sapphire glass and liquid nitrogen and then gently press against razor plate on a heating block and this temperature difference usually like lifts off the sapphire glass without any problems and we can then nicely use the very flat surface of the specimen for screening for the cells. And because Aaron wanted to know how this looks like so this is how our heating block looks like it's a Reishardt multi-plate from Leica and then there's a razor blade like glued with tape on top on the front and then we just press and then this releases usually the sapphire glass which you can see laying around here in large quantities. Okay and then because I told you that we use the histodide so we in the past used the safranin which was described by the paper from Roger but we found that the safranin gave not really consistent staining results so what I did was I looked in like histological books for all stains which can be used for staining nuclei and I came up with a list of 14 dyes and we tested all of them if they really stain if they like get can be dissolved in acetone and stain the nuclei or the DNA after embedded and being like in contact with heavy metals and being embedded in the epoxy resin.

 

And we came up with like four four dyes which worked really good crystal violet methyl green methylene blue and and of course the safranin and surprisingly some dyes did not work at all like ethidium bromide which I had high hopes for but for some reason they didn't give any fluorescent signal. Okay so what we then do is we put the specimen like upside down on a microscope immersed in water so we use the Leica SP5 and at 594 nanometer excitation light we really got nice signals from the cell specs and this is now like a view on the sapphire glass so you can even see the edge of the sapphire glass and you can see it's populated with a lot of cells and if you zoom in you can even detect different stages so this is the metaphase stage and anaphase stage maybe some interfaces so it's it's working very nicely actually and this is how how this um our four favorite dyes look like so our our favorite is really the methylene blue because it gives gives gives a very good signal to noise ratio a very reproducible staining and you can even like see single chromosome arms here and the same is true for the methyl methyl green and but it has a little bit more background background so you can see kind of the cytoplasm and if you're interested in like seeing that then maybe the methyl methyl green is better the safranin in principle works also still very nicely but as I said the staining reproducibility was varying and crystal violet I'm it works also nice but you can see that the chromosomes are not so distinctly visible as with the other dyes so we we decided for the methyl methylene blue and this is then how you can even do like a 3d scan of the embedded cell in the resin so you can even like segment like chromosome arms or single chromosomes in metaphase and like the important question was if if this interferes with outer structure but it does not so this is now a thin section of a neutral cell stained with methylene blue in the free substitution you can see that the chromosomes are not like kind of it doesn't show any precipitation and the microtubules are still very nicely visible so this will be published soon in this in the next clamp book of which thomas is editing okay so it says the same procedure as before that we have to trim them section them in 300 to 350 nanometer thickness and collect the sections and then of course we do also some post staining so we use 10 minutes of two percent uranyl and five minutes with a lead hydrate and then very importantly we have to add gold reducer so we just like put this gold reducers on on both sides of the grid and we need them later for the tomogram reconstruction okay and then we have to screen our sections and this we do to really select only the sections for tomography which are which contain the spindle and so this is just showing some examples of the of C. elegans embryo or this is now a male C. elegans which shows this meiotic cells in the center and this is now a U2AS cell and in all of them are some spindles right so here you can see the spindle in the U2AS cell this the male worm has full of cells of different stages so there it was really crucial to screen them to select only the region which was interesting for the tomography and the same as for the C. elegans embryo okay so once we have selected the sections we go to the Tecna F30 which you can see behind me so so we insert actually the sample in the beam and then we do two series so an A-stack and the B-stack we call it so what it means is we take a series of images of the same position of different tilts and then the sample is rotated by 90 degree and the same procedure is repeated and this is because in the electron tomography you have this intrinsic artifact called missing wedge it's because you cannot tilt more than 60 or 65 degree otherwise the sample thickness is too too high for the electron beam to penetrate and to reduce this missing wedge actually the we do this dual tilt approach and this reduces the missing image information to only missing cone but it's still there but we we can deal with this later in the image processing so we can still find our structures of interest. Okay once we have done the tilt acquisition we use the IMOS software which was developed by the Boulder lab of David Mastronardi and we based the image reconstruction on this fiducial marks of this gold fiducials which are on both sides of the of the plastic and we have to like trace and detect and trace the all the fiducials and all of the tilts and then use an algorithm called weighted back projection to actually get back the full volume of this section of the image information intersection in 3D and this is now how the sample it looks like after reconstruction.

 

I know it's a little hard to see but this is actually the full volume and I will show you the zoomed information of this later. Okay after after this was reconstructed we go to another software which is called Amira and I think for scientific collaborations there's this zip edition which and I think there's also a commercial Amira version the thing is this is Amira zip edition it's always like updated constantly of the week I would say so you can because we have an active collaboration with them they usually if we request some tools or some developments they put it in the new release and then we can always use it it's very nice. So what has to be done is we put the image data in and then we calculate the cylinder correlation so what that means is we search for an idealized cylinder of a certain diameter which is a microtubule.

 

So what it does is it transforms the image information to Fourier space and then looks for an idealized cylinder in Fourier space which is actually a bright dark bright signal with like a bright edge and then no I mean a bright search box and then the cylinder will be like a dark ring of course because the microtubule boundary is dark and then in the lumen of the microtubule it's bright again so that's what what it's actually looking for and then it looks in all possible orientations so this is done automatically and then after this is done we end up with two files which is a correlation and an orientation field and this field can then be searched for the microtubules and so we trace the microtubules automatically. So this is just to illustrate the whole process so what you see here is the central zones and those are the two pronuclei and it's the elegant embryo you can kind of see microtubules here but you don't want to trace them by hand of course and after calculating this correlation field what you can see now as a bright signal here is where there's a high correlation with our idealized cylinders so where the actually microtubule matches with the image information and then we can like search for the microtubules automatically based on our parameters like of a certain continuous length and then this can be then traced and overlaid to the original image data. So this is how we trace automatically the microtubules.

 

And of course no algorithm is perfect so there has to be like some manual correction so we have to delete false positives and also sometimes add some manual segmentations of microtubules which were obviously there but missed by the algorithm. And this is then how the final product would look like so this is the microtubule model of this section. And as our whole cell is much bigger than one section we have to repeat this.

 

So we combine, so we trace microtubules in all sections we selected beforehand and then this microtubules which actually cross the borders from some section to section we can use as a landmark. So we can use those to stitch the volumes together and afterwards there has to be a manual correction again to check if all connections have been found. And in the end we end up with a stitched volume with all the microtubules in and I just want to show you this model of a meromerodic spindle now so you can see chromosomes here and here and this is the X chromosome and you see it's full of organelles and mitochondria but this is how like the spindle is looking so it's