By analyzing cell cultures of the cone-like photoreceptor cell line 661W via SEM, we found two MNTs (MNT#1 and MNT#2) that clearly showed multiple bulges along their axis (see Fig. 1A–E). MNT#1 had a total length of 61.60 µm and MNT#2 of 13.74 µm.
The MNT bulge diameter (DB) was statistically significant larger for both MNTs compared to the MNTs diameters (DMNT) determined on bulge-free parts of the MNTs (median, 95% confidence interval (CI)); DB#1 = 247.5 (220.0, 440.0) nm (n = 6) vs. DMNT#1 = 110.0 (55.0, 220.0) nm (n = 100), Bayes factor (BF) >100, effects size (Cohen’s d): d = -4.389; DB#2 = 85.0 (68.0, 136.0) nm (n = 4) vs. DMNT#2 = 68.0 (51.0, 102.0) nm (n = 100), BF = 23.4, d = 1.609) (see Fig. 1G). This analysis proofs that the visually observed bulges are real deformations of the MNT membrane. The analysis also shows that the larger the diameter of the MNTs is, the larger is the diameter of the MNT bulges (as quantified by the larger absolute differences of the medians (Δm) of DB values of MNT#1 (Δm = 137.5 nm, increase of 225%) compared to MNT#2 (Δm = 17.0 nm, increase of 125%)).
The distance between successive MNT bulges (dBB) was dBB = 7.527 (4.890, 9.451) µm for MNT#1 (n = 5) and dBB = 2.492 (1.644, 3.610) µm for MNT#2 (n = 3). The difference was statistically significant (BF = 11.27, d = 3.041) (see Fig. 1H).
The length of the MNT bulges (LB) was LB = 1.700 (1.536, 2.742) µm for MNT#1 (n = 6) and LB = 0.3495 (0.2730, 0.4260) µm for MNT#2 (n = 4). The difference was statistically significant (BF = 95.83, d = 3.879) (see Fig. 1H).
Our finding of the presence of bulges on MNTs is in agreement with previously published studies with other cell types. Wittig et al. documented examples of MNT bulges in the retinal pigment epithelial cells using differential interference contrast microscopy and SEM. The MNT bulge diameter was reported to be DB = 1 µm and the typical MNT diameter to be DMNT = 250 nm (with a range of 50–300 nm). These values are larger than the values we determined in our sample and study, i.e. the diameter of the two MNTs investigated in our study were 2.3 (MNT#1) and 3.6 (MNT#2) times smaller, and the bulge diameters were 4.04 (MNT#1) and 11.75 (MNT#2) times smaller, respectively. This indicates the presence of a large heterogeneity of MNT morphology, confirming previous reports. Wittig et al. concluded that the MNT bulges suggest the presence of organelles in MNTs. Using the specific mitochondrial dye JC-1 they could label mitochondria inside MNTs. The presence of mitochondria inside MNT bulges was also reported by Patheja and Sahu. In another study, Reichert et al. reported also the presence of MNT bulges between cells (human primary CD34+ haematopoietic progenitor cells and leukaemic KG1a cells). The MNT bulge thickness was described to be smaller than 100 nm in diameter, in agreement with our finding concerning MNT#2.
With our study, we were the first to analyze LB and dBB values of MNT bulges. The range of LB values obtained (0.2730–2.742 µm) overlaps with the length distribution of mitochondria in cells (e.g. in retinal pigment epithelial ARPE-19 cells: 0.4–74 µm, primary cortical neurons: 1.27 ± 0.04 µm) as well as with the size distribution of peroxisomes (0.1–1 µm). The length of mitochondria is, however, a variable parameter that depends not only on the specific cell type but also on the state of the cell, i.e. cell cycle or metabolic state.
That the location of MNT bulges was seemingly not random on the MNT is a novel finding of our study. The distance values between successive MNT bulges, as quantified by dBB, showed unimodal distributions for both MNTs investigated. The ratio dBB/LB based on the median values was 4.43 for MNT#1 and 7.13 for MNT#2, respectively; and the ratio dBB/DB based on the median values was 68.4 for MNT#1 and 25 for MNT#2, respectively. This indicates that the periodicity of the bulges on an MNT seems to be independent of the MNT diameter as well as the length of the MNT bulges. Concerning the cause of the MNT bulge periodicity, no clear mechanism can be envisaged. Either it is a finding due to chance, or there is an underlying unexplored process that regulates the distance between successive bulges of MNTs. It could be indeed that it is only a finding due to chance since only a limited number of data points (i.e. distance values) were available for the analysis. Further studies are needed to investigate this aspect with a larger sample size.
What could be in general the cause of the MNT bulges observed? We think that three possible causes should be considered: (1) MNT bulges as artefacts caused by the cell staining process involving the SEM analysis, (2) MNT bulges as local deformations of the MNT without the presence of a cargo inside, or (3) MNT bulges as effects of an object inside the MNT that deforms the enclosing MNT locally.
Explanation (1) is unlikely since MNT bulges were also observed by optical microscopy, and MNT bulges are only occasionally observed (contrary to the expectation of the MNT bulge formation being a results of a general SEM staining artefact that should affect all or a large part of all MNTs present in the investigated culture). Explanation (2) is in principle possible but there are no nanomechanical processes known yet that result in an oval local deformation of an MNT without the involvement of an object inside the MNT causing the deformation. Explanation (3) is most likely since (i) the presence of mitochondria inside MNT bulges was previously shown by several studies; (ii) the MNT bulge diameter values determined in our study (range: 68.0–440.0 nm) correspond to diameters of organelles like mitochondria (typical diameter: 100–1000 nm), peroxisomes (typical diameter in RPE cells: 100–300 nm), or exosomes (typical diameter: 30–100 nm); (iii) the length of the MNT bulges overlap with the length distributions of mitochondria and peroxisomes, and (iv) movements of MNT bulges were reported by other studies (speed: 0.16 µm/s, 20.7 ± 2.3 µm/h, 0.08 µm/s, 0.0259 ± 0.0079 µm/s, 0.033–0.059 µm/s, 0.045 ± 0.005 µm/s) indicating the presence of a real object inside the MNT.