Category Archives: Layered intracisternal protein granules in mammalian lung

For 35 years I have thought about this protein organized so nicely in the type II cells of the lung of three mammalian species (guinea pig, ferret, mongrel dog) which I am sure is present in more species, and have not had the time to investigate it further. A peer review publication in the early 1980s was about as far as the study went, though published in an OK journal (Ultrastructure Research) I felt there was more to see than just what I could put down in those few pages. Life makes us make choices, and at this point I choose to play with this old problem. I am not professing anything except diligent “musings” here.

100 nm square = alveolar type II cell granule pattern

Electron microgaphs of lung, Layered intracisternal protein granules in mammalian lung, Ultimate order, the cell

A summary and diagram – two years in the making (not so LOL)

A protein granule exists in the rough endoplasmic reticulum of alveolar type II cells of some species which has remained relatively obscure. It has characteristics of a highly self-associated protein with a predictable repeating substructure and displays at least 8 prominent patterns:

1) most commonly it appears as a layered sheet about 100 nm thick
2) which upon cross section comprises two dense outer layers, 100 nm apart, a less dense central layer which can have up to 3 faint layers on either side. This pattern forms one period
3) the layered sheet (100 nm-thick period) can also be stacked many times creating a granule with a length and height of many microns
4) whether single or highly stacked, ribosomes only lie on growing ends of the granule and in opportune cuts a single period has 4 ribosomes, 1 at each dense layer and one on either side of the central layer
5) the perimeter of the granule could be linear, or rounded, with sheets (layers) arranged in patterns reminiscent of flow extrusion, where each of the 4 ribosomes equates to a die, and as if the rate of protein translation/modification, and the shape, size and press capacity of the ER determined if the granule would be gently curved or U shape, looped or folded-back, concentric, or branched. Some granules were a mixture of all these forms, even rarely intersecting
6) Both the outer dense, and less dense central layers appeared to be continuous on perpendicular cuts, but clearly punctate when cut tangentially
7) Outer dense bands showed a periodicity of about 4 per 100 nm, while less dense central bands showed a periodicity of about 5-7 per 100 nm, possibly lined up in a staggered format
8)  A faint vertical tie was also seen at about 4 per 100 nm beginning typically at one of the densities

Measurements have been made but these are approximations derived from hundreds of micrographs and numerous publications. Similarly, measurements by numerous reports on the size and structure of surfactant protein A have shown that it is likely to be an 18-mer with a spread of the CRD at the top of the bouquet of something on the order of 25 nm. It doesn’t take a big leap to put four 18-mers together vertically and copy them side to side to come out with a pretty awesome banding pattern that fits the 100 nm dimensions.

most_up_2_date_type_II_cell_granule_diagram

Single period alveolar type II cell granule: end-on

Electron microgaphs of lung, Layered intracisternal protein granules in mammalian lung, The cell: images, Ultimate order, the cell

Electron micrograph of an alveolar type Ii cell which is overrun with surfactant protein granules and an isolated part of a larger granule shows this profile which is so symmetrical as to look like the spokes of a wheel. Micrograph on left is unretouched, micrograph on the right is burned in the areas of periodicity of the molecules of surfactant protein, and dodged in the areas between, just slightly to highlight what I saw.

The purpose of the study is still to determine which of the surfactant proteins is responsible for forming the granules. So here is such a single period of a granule which has a ribosome marked for approximate size (red dot=27 nm) red bar=100 nm superimposed on an electron micrograph (unretouched). Right hand image has emphasized image, and a circumference of the round less-dense inner band of the period (central dense dot represents the “outer dense band” seen in linear profiles. The inner dots have been counted in linear granules at closer to 5-6 per 100 nm but here there are 11 or 12 dots of the less dense central band under the circumference, which is about 330 nm, or a little less, and in this photo the dots measure about 15-17 nm (consistent with previous measurements, then are spaced at about 25 nm apart. Not everything lines up exactly with previous posts on this subject in the linear profiles, but clearly they are analogous and match pretty well.  The spoke appearance likely corresponds to the vertical lineup of molecules seen in granules that have a linear arrangement. (just as an aside, there is something spooky about the fact that I have found several times now that the space between these molecules in the central less dense band are spaced apart the same distance as a ribosome…. that is just uncanny, and I bet it will end up meaning something about protein synthesis in general–just a heads up).  see this post and this post

6758_17082_gpig_301_single_circle

Guinea pig alveolar type II cell granule protein: surfactant protein A?

Electron microgaphs of lung, Layered intracisternal protein granules in mammalian lung, The cell: images, Ultimate order, the cell

Guinea pig alveolar type II cell granule protein: surfactant protein A?

There are so many images which show the same data, layering, periodicities, hexagonal array at the growing ends of granules that the evidence that these are some regular sort of structure in select species is a little hard to ignore. I keep trying to convince myself that I am seeing what I think I am seeing.  This opportune section of an alveolar type II cell (from ferret) granule is just kind of nice, as it shows the growing end ribosomes, the stiffness of the RER profile when the protein begins to organize (seen on the left hand side of the micrograph) the looseness at the growing end before further oligomerization takes place in the granule (right hand side of the micrograph)  where it seems that there is some kind of organization soon after the protein leaves the ribosomes (like the hexagonal pattern for a top down view of surfactant protein A, as seen here (unretouched EXCEPT for the burn tool in photoshop to highlight what I have seen), though I would have liked to see more with the central dot.  Ribosomes were burned to highlight, and a ribosome marker (nominal 27 nm) are given plus a bar marker at approximately 100 nm, and hexagons at the growing end of the granule are burned as well.  Inset is enlarged from white box in top figure. 9835_17084_guinea_pig_#301

9778_31065_ferret_type_II_granule9778_31065_ferret_type_II_gr_inset

More on two levels of periodicity in alveolar type II cell granules: guinea pig

Electron microgaphs of lung, Layered intracisternal protein granules in mammalian lung, Ultimate order, the cell

Below are some examples of an alveolar type II cell granule in an electron micrograph from a guinea pig lung which I am showing unretouched, and then enlarged, and same image with periodicity enhanced with the burn tool in photoshop, enlarged again and nanometer markers included in each increased magnification.

From the four figures below, it seems that the relative size difference in the periodicities and the number per nanometer is similar with dog.

Fig 1 (top) is from a guinea pig alveolar type II cell. Bar = 100 nm. Box is an area enlarged to become Fig 2, seen just below Fig. 1, which is an unretouched electron micrograph of a single granule.  It is easy to see the periodicity in the outer dark band and the lighter central band is different. This view is just a slightly tangential cut to the long axis of the granule. Figures 1-part of 5 are from 9835_17084_gpig_anm#301.

Fig. 3 shows the same image as Fig. 2, except that I have burned the densities (periodicities) in the middle less dense band (smaller dots) and the outer dense band (larger dots) so they are more clearly visible.

And in Fig. 4, the box indicated in Fig. 3 is rotated to show the periodicities and nanometer marks from a ribosome, the whole band-period of 100 nm, the periodicity and approximate diameter of the proteins comprising the smaller dots (approximately 15-17 nm in diameter seen as the purple dots), at about 5-6 dots per 100 nm, per the bar marker.

The next figure (Fig 5.) compares guinea pig (on the right) with dog, in a similar assessment of the middle band periodicities (dog on the left).  NB, the larger periodicities (from the outer dense bands of one basic period of this granule (100 nm width typically) are clearly seen in guinea pig images, but only slightly recognizable, find maybe two at most, in the image of dog.

9835_17084_gpig_periodicity-19835_17084_gpig_periodicity-29835_17084_gpig_periodicity-3gpig_dog_middle_line_periodicity_comparison

Tangential cuts through alveolar type II cell granules show dense layer substructure

Electron microgaphs of lung, Layered intracisternal protein granules in mammalian lung, The cell: images, Ultimate order, the cell

Tangential cuts through alveolar type II cell granules show dense outer layers of this regularly arranged molecular substructure, has a periodicity to it, albeit densities of a different size than at other sites in the , just like the less dense middle band of these granules.  Images below also show a slight tendency for vertical banding, within the horizontal layers of the surfactant protein (A ? ) granule. The hierarchy for all the level of substructural organization are here:

  1. 100 nm period (height) (no restriction on length of granule seen) which has 1,3,5 or 7 layers between the outer dense bands
  2. Outer dense layers on perpendicular cut look continuous, on tangential cut however, they show a periodicity with large round densities about 2.5 per 100 nm length.
  3.  Moderately dense central band shows a periodicity of 5-7 densities per 100 nm
  4. vertical stripes, a kind of banding, perpendicular to the long orientation of mos that coinciding with the periodicity of the central dense  band.

Negative 7088_23356)ferret_#17more_vertical_banding-1more_vertical_banding-2more_vertical_banding-3

Vertical and middle layer organization in alveolar type II cell surfactant protein granules

Electron microgaphs of lung, Layered intracisternal protein granules in mammalian lung, The cell: images, Ultimate order, the cell

Vertical and middle band banding in alveolar type II cell surfactant protein granules is shown here (hopefully this is SP-A). Using the same image as posted as the quintessential surfactant protein granule in alveolar type II cells, the image has been copied, inverted, and offset just a few pixels, to cast a shadow and give dimension to the “edges” in the image, a kind of embossing technique.

Embossing vertically highlights the bumpiness of ribosomes and other protein “lumps” within the granule, while embossing by moving the inverted image below, causes the ribosomes to appear to be concave.  Both accentuate different features (see below). The embossing upward as well as moving the inverted image a few pixels to the left highlight the banding seen within the granule which is secondary to the electron dense bands that run parallel to the long dimension of the granule… the vertical lines are not present in the surrounding cytoplasm, thereby indicating that the vertically lines within the granule are not section artifact or fixation artifact but actually a part of the specific alignment of the granule proteins.  The third image is a flattened image, cropped, increased in brightness and contrast, then vertical lines were superimposed where I saw definition.  Ribosomes, as an internal control for magnification are about the size of the red marker (27 nm estimated diameter), black line of 300 nm = the height of the three banding periods comprising this particular granule, 100 nm bars mark out the middle dense layer (with the blue dots where periodicity shows up) and also shows that about 4-5 protein densities occur in the middle layer of each 100 nm period.

Original ferret alveolar type II cell RER granule electron micrograph, untouched, from which these processed images were derived can be found here.

Negative 9879 block 23494 ferret # 16, untreated. Electron micrograph of an alveolar type II cell with a surfactant protein granule (which in some ways is similar to other collectin, c-type lectin, style granules, where organization of these proteins is highly oligomerized–e,g, Birbeck granules).  Embossed upward, and down ward images below. Bottom image is a cropped and enlarged from middle image, showing vertical lines which may be 4 or 5 per 100 nm and can transcend all three periods of this granule; ribosomes (as a size marker of @27 nm); periodicity of the medium dense layer of this granule (blue dots and 100 nm bar marker).

quintessential_SP_granule_shadowed-1quintessential_SP_granule_shadowed-2vertical-banding

 

 

 

Alveolar type II cell granule in ferret – electron microscopy

Electron microgaphs of lung, Layered intracisternal protein granules in mammalian lung, Ultimate order, the cell

More on the periodicity of the dense outer bands of the alveolar type II cell surfactant granule.  Still think this is probably surfactant protein A. I have been assessing the densities in the darker outer bands, which are rounded molecular complexes, much more clearly seen on the tangential plane (yesterday’s images), and which looks to be something over 30 nm in diameter (using an adjacent ribosome as the size reference), and only two (with space between) appear in any 100 nm stretch.  The punctate nature of the lighter bands in this granule seemed to be more like 5-7 per 100 nm, so there is an obvious difference, both in size and in spacing of these two entities. How do they knit together into the granule?.

Dense bands are more continuous than dotted, particularly views perpendicular to the length of banding, but I would suspect that differences in the appearance of dense band periodicity has to do with the way the proteins denature depending upon the constituents of the fixation fluid, whether more or less calcium is present, or whether it is more hypertonic, or isoosmolar, but this is conjecture.

Figure below shows the intermittent densities in a perpendicular cut, unlike the post yesterday which showed the dense band densities on a tangential cut (and more pronounced). Figures below are arranged thus:

  1. quintessential alveolar type II cell surfactant protein granule UNRETOUCHED- Electron micrograph shows a granule with three linear periods and a ballooned end. Diameter of one ribosome is taken to be 27 nm, one period 100 nm (vertical black bar marker) therefore the whole granule here is approximately 300 nm in height (thickness). TOP
  2. quintessential alveolar type II cell surfacant protein granule with ribosomes which are on growing ends (and in this particular image, on the balloon end, and a couple of ribosomes on the opposite end, with membrane without ribosomes parallel to the long axis of banding. Ribosomes are PINK – and – the
  3. quintessential alveolar type II cell surfactant protein granule itself, is BLUE
  4. measurements of periodicity in dense as well as light bands BOTTOM, with green line over green dots (representing about 15 nm diameter punctate periodicity for lighter bands and red line over red dots (30+ nm diameter) showing periodicity of dense band protein molecules.

quintessential_SP_granulequintessential_SP_granule_overlay-1quintessential_SP_granule_overlay-2

Alveolar type II cell intra-RER protein organization: ferret, more hexagonal structures

Electron microgaphs of lung, Layered intracisternal protein granules in mammalian lung, The cell: images, Ultimate order, the cell

Alveolar type II cell intra-RER protein organization: ferret, more hexagonal structures per the last two posts, there is more order here,  actual linear densities perpendicular to the long axis of the layered granule, and these blend out into the tangential plane as hexagonal structures with a central dense area (either 1 or 2 dots — in this case mostly 1 dot).

Top electron microgr9855_23349_ferret_17aph (unretouched, except to show the bounding box of the inset image) is an enlargement from a negative (9855) of a ferret type II cell, of just one of the layered granules present which is particularly well suited to examination of its tangential-orientation and detection of patterns that might indicate that it is surfactant protein A.

Bottom electron micrograph is inset, enlarged, and the patterning perpendicular to the long axis of the granule layering (here, tangentially cut) is seen, and the erase tool has been used to outline what could be hexagonal patterns for the surfactant protein A 18-mer bouquet.  You are free to decide if this looks right. My question is that they are small, but also would represent just 1/4 of the 100 nm width of the real pattern-period, of mirrored and stacked (4 molecules) of SP-A 18-mers. I have not highlighted all the vertical lines, nor have i highlighted all the hexagonal structures.

9855_23349_ferret_17-inset

 

 

 

 

Alveolar type II cell intra-RER protein organization: ferret

Electron microgaphs of lung, Layered intracisternal protein granules in mammalian lung, The cell: images, Ultimate order, the cell

hexagonal_proteins_in_alveolar_type_II_cellsMore tangential sections of RER layered protein from ferret alveolar type II cells seen with electron microscopy. I have chosen one of the more easily identifiable granules with the light portion of the banding ‘periods’ spread out so that any order seen on from the “top down” might be highlighted.  I have outlined in red (this is a “partial erase” in photoshop, against a red (and green) layer), no other manipulation of the micrograph has occurred.

The red outlines are pretty obvious hexagonal structures, seemingly mostly with a central dense area, but way to often for chance, two central dots.  I have seen this so many times that it becomes necessary to call attention to it, and likely it means something in the organization of this surfactant protein (which I am calling surfactant protein A). On the other hand, the size of a ribosomes in these micrographs would make the hexagons a little too small….  maybe only 75- 80 nm across, so this is an issue to reconcile.

Red outlines, within the tangentially spread inbetween layers), green outlines, an area which might be still within this particular RER profile, but might actually be cytoplasmic.  I have compared the incidence of cytoplasmic heaxagonal structures, there are some, no question, and I don’t want to read more into the hexagonal molecular organization than is warranted.  To me however, it is significantly greater within the membrane of the RER when tangentially sectioned as happened here, and the previous post (same animal, same micrograph, same magnification, different site.

 

Factors that may influence granule layering patterns in alveolar type II cells

Electron microgaphs of lung, Layered intracisternal protein granules in mammalian lung, The cell: images, Ultimate order, the cell

The granule in the alveolar type II cell of guinea pig, ferret and dog is unique, in that it appears to have wonderful shapes, concentric, U-shaped, linear, branching (even highly branching mostly at perpendicular planes to the original layering). I was trying to figure out what could cause such variation in shape. One keeps in mind that these are cross-sectional images, 2D of the 3D cytoplasmic granules, and therefore can be difficult to interpret as a whole granule.

To note first: there are often parallel “jets” (which I have called periods, just to be proper ) for the production of surfactant protein A (yep, I am calling it that with nothing but circumstantial evidence). Sometimes there are 10 or 15 such nidi of production, each perpendicular to the long axis of the layering (banding) and having about 4 ribosomes (the 4th ribosome in each period serves as the 1st in the adjacent layer).  The amount of resistance at the opposite end of the granule to protein synthesis, and the rate of protein synthesis (at the growing end(s) of the granule where the ribosomes are ) will determine whether this “jet” will spew out SP-A in an unhindered stream to create a linear granule or whether it will buckle, twist, spin or curl.

I began searching extrusion images on google, and these below (easily-found set of 6   extrusion images which illustrate the point) do a really good job of matching what is seen electron microscopically, of what becomes of the structure of protein layering during SP-A production when the protein produced meets with resistance.  Awesome… ha ha, a natural physical phenomenon, I will bet. What scientific journal will publish my diagram… ha ha, probably not.  Too funny.

extruded_soft_serve_polyethylene_pasta_model