Your browser is out-of-date!

Update your browser to view this website correctly. Update my browser now

×

Discipline
Biological
Keywords
Membrane Nanotubes
Tunnelling Nanotubes
Ultrastructure
Observation Type
Standalone
Nature
Standard Data
Submitted
Feb 1st, 2018
Published
Mar 7th, 2018
  • Abstract

    Membrane nanotubes (MNTs) are nanotubular cell-to-cell connections enabling cell-to-cell signaling and cargo transfer. The presence of local MNT bulges has been reported by several studies. However, a detailed characterization of MNT bulges concerning their geometrical properties has not been done yet. The aim of our study was to analyze MNT from cone-like photoreceptor cells (661 W) using scanning electron microscopy (SEM) in order to characterize MNT bulges, thereby increasing the knowledge of ultrastructural feature of MNTs. Our SEM analysis of two MNTs with multiple bulges revealed that (i) MNT bulges are characterized by a statistically significant local increase in the MNT diameter (125% and 250% for both MNTs analyzed), (ii) the thickness and length of the MNT bulges correspond to dimensions of mitochondria, peroxisomes and exosomes, and (iii) the MNT bulges seem not to be randomly distributed on the MNTs but exhibit a preferred spacing with a different median value for each MNT. Our findings highlight that the ultrastructure of MNT exhibits interesting properties that need to be further investigated.

  • Figure
  • Introduction

    Nanotubular cell-to-cell connections termed “tunnelling nanotubes” or “membrane nanotubes” (MNTs) (the term used in this paper) enable diverse possibilities of cell-to-cell signaling and cargo transfer. This includes the exchange of signal carriers (e.g. proteins), organelles (e.g. mitochondria), bacteria, viruses, exosomes, DNA, RNA, or electric long-range coupling.

    The defining characteristics of MNTs are still being debated and the investigation of MNT properties is ongoing but recently a consensus of leading MNT researchers was published that MNTs can be defined as “tubular membrane connection between non-adjacent cells that allow direct intercellular communication, not necessarily gap junction-mediated”. MNTs also contain F-actin and/or tubulin, have a variable diameter of 50–800 nm and are open-ended. It can be added that MNTs are filled with cytoplasm and have a lipid bilayer. Some MNTs also contain microtubules and have the gap junction protein Cx43 at the end. In some cases, individual MNTs stick together to form a single, thicker, MNT.

    As recently summarized by Rustom, MNTs are “intimately linked to the physiological state and pathological” state of cells and “represent a central joint element of diverse diseases, such as neurodegenerative disorders, diabetes or cancer”. Furthermore, MNTs seem to play an important role in long-range physical cell-to-cell signaling in multicellular organisms possibly enabling novel ways of physical signal transfer and being involved in the functioning of neurosystems.

    Studies have been published that report the existence of bulges, i.e. local increases of the diameter, of MNTs. This ultrastructural feature of MNTs was attributed to the presence of objects (vesicles or organelles) inside MNTs.

  • Objective

    Our objective was to document and analyze bulges of MNTs using scanning electron microscopy (SEM) to increase the knowledge of ultrastructural feature of MNTs. To our knowledge, a detailed characterization of MNT bulges concerning their geometrical properties has not been done before.

  • Results & Discussion

    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#1m = 137.5 nm, increase of 225%) compared to MNT#2m = 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.

  • Conclusions

    Our SEM analysis of two MNTs from cone-like photoreceptor cells of the cell line 661W with multiple bulges revealed that (i) bulges are characterized by a statistically significant local increase in MNT diameter (125% and 225% for both MNTs investigated); (ii) the thickness and length of the MNT bulges correspond to dimensions of mitochondria, peroxisomes and exosomes, and (iii) the MNT bulges seem not to be randomly distributed on the MNTs but exhibit preferred spacing with a different median value for each MNT. Our findings shed new light on the microstructure of MNTs and MNT bulges in particular.

    Further studies should replicate and extend our findings with more MNTs and by applying SEM and fluorescence microscopy simultaneously by correlative light and electron microscopy (CLEM) or even better 3D CLEM with serial blockface (SBF) SEM, as recently developed.

  • Limitations

    There are two main limitations in this study. First, only two MNTs could be identified in the SEM images taken from multiple sections of two cell cultures, resulting in a small number of MNT bulges that could be used for the statistical analysis of their geometrical properties. Secondly, we did not employ fluorescence microscopy to a nalyzewhether the MNT bulges were colocalized with the presence of fluorescence markers, e.g. JC-1 indicating the mitochondria. This, however, could not be performed for technical reasons: performing SEM and fluorescence microscopy analysis of the same MNT consecutively with two instruments (as available in our lab) is nearly impossible due to change and disturbance of the cell/MNT state caused by the transportation between microscopes and the time-delay involved processing the sample for SEM analysis causing changes in the MNT state. A CLEM analysis would have been a solution.

  • Methods

    Cell culture

    Mouse cone-like photoreceptor cells 661W (University of Oklahoma, Oklahoma City, OK) were maintained and treated in DMEM (Gibco, Life Technologies, USA) supplemented with 10% FCS (Biochrom, Germany). The cells were grown at 37°C in a humidified environment with 5% CO2 and passaged every 3 to 4 days. For experimental procedures, cells were briefly rinsed with PBS (DPBS, Gibco, Life Technologies, USA). Afterwards, 1 ml of 0.25/0.02% (w/v) of trypsin in EDTA solution (Biochrom, Germany) was added to the cell culture and they were incubated at 37°C and with 5% CO2 for 5 min. Trypsin activity was neutralized by adding medium and centrifugation at 150 g for 5 min.

    Scanning electron microscopy

    For the scanning electron microscopy (SEM) analysis, cells with a cell number of 6×104 were grown on 12 mm round glass coverslips for 24 h. They were fixed with modified 1% glutaraldehyde in 100 mM a phosphate buffer (DPBS, Gibco, Life Technologies, USA). After several washes in PBS, the samples were postfixed in 1% osmium tetroxide/PBS for 2 h on ice, washed in water, en bloc contrasted in 1% uranyl acetate/water, and dehydrated in a graded series of ethanol (30%, 50%, 70%, 90%, 96%, 2×100% ethanol; pure ethanol on molecular sieve). Finally, they were critical point dried using the Leica CPD 300 (Leica Microsystems, Vienna, Austria) and mounted on 12 mm aluminium stubs. Cells mounted on pieces of plastic were glued to conductive carbon pads and the edges of the plastic were surrounded with silver paint to reduce charging. Mounted samples were sputter coated with gold using the Baltec SCD 050 (Leica Microsystems, Vienna, Austria), and analyzed with a Jeol JSM 7500F cold field emission SEM (Jeol, Eching, Germany) at 8 mm working distance and 5 kV acceleration voltage using the lower secondary electron detector.

    SEM image analysis has been performed in ImageJ (NIH). 78 SEM images were obtained and manually screened for MNT bulges in total. Four parameters were determined for further analysis of the MNT ultrastructure: the MNT diameter, DMNT, the diameter of the bulge, DB, the distance between adjacent bulges, dBB, and the length of the bulges LB. LB was determined by fitting an oval to the MNT bulge and determining the length from one end of the oval to the other.

    Statistical analysis was conducted with JASP (https://jasp-stats.org). For comparison of distributions, the Bayesian independent samples t-test with a Cauchy scale of 0.5 was used. A Bayes factor of >10 was assigned as a threshold for a statistically significant difference of the distributions. Figure 1 was created using JASP and Adobe Illustrator.

  • Acknowledgements

    The author would like to thank Dr. Thomas Kurth (TU Dresden, Center for Regenerative Therapies Dresden) for realization of electron microscopy and Simon Schaefer for proofreading the manuscript.

  • Ethics statement

    Not applicable.

  • References
  • 1
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    2
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    3
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    4
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    5
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    Matters12.5/20

    Ultrastructural features (bulges) of membrane nanotubes between cone-like photoreceptor cells: An investigation employing scanning electron microscopy

    Affiliation listing not available.
    Abstractlink

    Membrane nanotubes (MNTs) are nanotubular cell-to-cell connections enabling cell-to-cell signaling and cargo transfer. The presence of local MNT bulges has been reported by several studies. However, a detailed characterization of MNT bulges concerning their geometrical properties has not been done yet. The aim of our study was to analyze MNT from cone-like photoreceptor cells (661 W) using scanning electron microscopy (SEM) in order to characterize MNT bulges, thereby increasing the knowledge of ultrastructural feature of MNTs. Our SEM analysis of two MNTs with multiple bulges revealed that (i) MNT bulges are characterized by a statistically significant local increase in the MNT diameter (125% and 250% for both MNTs analyzed), (ii) the thickness and length of the MNT bulges correspond to dimensions of mitochondria, peroxisomes and exosomes, and (iii) the MNT bulges seem not to be randomly distributed on the MNTs but exhibit a preferred spacing with a different median value for each MNT. Our findings highlight that the ultrastructure of MNT exhibits interesting properties that need to be further investigated.

    Figurelink

    Figure 1. Scanning electron microscopy (in lower secondary electron image mode) images of bulges of MNT between cone-like photoreceptor cells (661W).

    (A, C) Two MNTs (MNT#1 and MNT#2) with bulges (indicated with red arrows). Scale bar of image A: 10 µm. Scale bar of image C: 1 µm. In figure C, a single bulge of a third MNT is indicated with a yellow arrow. The two MNTs are connected at one side with the cell and on the other side unconnected. The disconnection seems to be the result of the SEM staining process, possibly disrupting the state of the MNTs causing the breakage of long MNTs in some cases.

    (B) Magnification of a MNT bulge selected from image A.

    (D, E) Magnification of two MNT bulges selected from image C. In D the bulge is clearly visible where in E it is more difficult to recognize the bulge.

    (F) Diagrammatic drawing of an MNT with two bulges and the four parameters determined for further analysis: MNT diameter (DMNT), diameter of the bulge (DB), distance between adjacent bulges (dBB) and the length of the bulges (LB). In the lower visualized MNT bulge in F an oval is shown (red) that was used to determine LB.

    (G, H) MNT diameter and bulge diameter values, as well as, distance between adjacent bulges for both MNTs. Statistically significant (Bayer factor >10, Bayesian independent samples t-test) differences are indicated (*).

    It should be noted that MNT#1 is approximately twice as wide as MNT#2. Smaller cargo in MNT#1 might therefore not be visible.

    Introductionlink

    Nanotubular cell-to-cell connections termed “tunnelling nanotubes” or “membrane nanotubes” (MNTs) (the term used in this paper) enable diverse possibilities of cell-to-cell signaling and cargo transfer[1][2][3]. This includes the exchange of signal carriers (e.g. proteins), organelles (e.g. mitochondria), bacteria, viruses, exosomes, DNA, RNA, or electric long-range coupling[4][5][6][7][8].

    The defining characteristics of MNTs are still being debated and the investigation of MNT properties is ongoing but recently a consensus of leading MNT researchers was published that MNTs can be defined as “tubular membrane connection between non-adjacent cells that allow direct intercellular communication, not necessarily gap junction-mediated”. MNTs also contain F-actin and/or tubulin, have a variable diameter of 50–800 nm and are open-ended[3]. It can be added that MNTs are filled with cytoplasm and have a lipid bilayer[9]. Some MNTs also contain microtubules[10] and have the gap junction protein Cx43 at the end[6]. In some cases, individual MNTs stick together to form a single, thicker, MNT[9].

    As recently summarized by Rustom[11], MNTs are “intimately linked to the physiological state and pathological” state of cells and “represent a central joint element of diverse diseases, such as neurodegenerative disorders, diabetes or cancer”. Furthermore, MNTs seem to play an important role in long-range physical cell-to-cell signaling in multicellular organisms possibly enabling novel ways of physical signal transfer[12] and being involved in the functioning of neurosystems[13].

    Studies have been published that report the existence of bulges, i.e. local increases of the diameter, of MNTs[14][15][16][17][6][18][19][20][21][22]. This ultrastructural feature of MNTs was attributed to the presence of objects (vesicles or organelles) inside MNTs[6].

    Objectivelink

    Our objective was to document and analyze bulges of MNTs using scanning electron microscopy (SEM) to increase the knowledge of ultrastructural feature of MNTs. To our knowledge, a detailed characterization of MNT bulges concerning their geometrical properties has not been done before.

    Results & Discussionlink

    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#1m = 137.5 nm, increase of 225%) compared to MNT#2m = 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[14][15][6][20]. Wittig et al.[6] 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[22]. In another study, Reichert et al.[20] 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[23]: 0.4–74 µm, primary cortical neurons[24]: 1.27 ± 0.04 µm) as well as with the size distribution of peroxisomes (0.1–1 µm)[25]. 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[27].

    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[6], 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[28][17][6][29][30][18][19][22]; (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)[31][32][33], peroxisomes (typical diameter in RPE cells: 100–300 nm)[34], or exosomes (typical diameter: 30–100 nm)[35][36]; (iii) the length of the MNT bulges overlap with the length distributions of mitochondria and peroxisomes[23][34], and (iv) movements of MNT bulges were reported by other studies (speed: 0.16 µm/s[14], 20.7 ± 2.3 µm/h[37], 0.08 µm/s[19], 0.0259 ± 0.0079 µm/s[38], 0.033–0.059 µm/s[39], 0.045 ± 0.005 µm/s[21]) indicating the presence of a real object inside the MNT.

    Conclusionslink

    Our SEM analysis of two MNTs from cone-like photoreceptor cells of the cell line 661W with multiple bulges revealed that (i) bulges are characterized by a statistically significant local increase in MNT diameter (125% and 225% for both MNTs investigated); (ii) the thickness and length of the MNT bulges correspond to dimensions of mitochondria, peroxisomes and exosomes, and (iii) the MNT bulges seem not to be randomly distributed on the MNTs but exhibit preferred spacing with a different median value for each MNT. Our findings shed new light on the microstructure of MNTs and MNT bulges in particular.

    Further studies should replicate and extend our findings with more MNTs and by applying SEM and fluorescence microscopy simultaneously by correlative light and electron microscopy (CLEM)[40] or even better 3D CLEM with serial blockface (SBF) SEM, as recently developed[41].

    Limitationslink

    There are two main limitations in this study. First, only two MNTs could be identified in the SEM images taken from multiple sections of two cell cultures, resulting in a small number of MNT bulges that could be used for the statistical analysis of their geometrical properties. Secondly, we did not employ fluorescence microscopy to a nalyzewhether the MNT bulges were colocalized with the presence of fluorescence markers, e.g. JC-1 indicating the mitochondria. This, however, could not be performed for technical reasons: performing SEM and fluorescence microscopy analysis of the same MNT consecutively with two instruments (as available in our lab) is nearly impossible due to change and disturbance of the cell/MNT state caused by the transportation between microscopes and the time-delay involved processing the sample for SEM analysis causing changes in the MNT state. A CLEM analysis would have been a solution.

    Methodslink

    Cell culture

    Mouse cone-like photoreceptor cells 661W (University of Oklahoma, Oklahoma City, OK) were maintained and treated in DMEM (Gibco, Life Technologies, USA) supplemented with 10% FCS (Biochrom, Germany). The cells were grown at 37°C in a humidified environment with 5% CO2 and passaged every 3 to 4 days. For experimental procedures, cells were briefly rinsed with PBS (DPBS, Gibco, Life Technologies, USA). Afterwards, 1 ml of 0.25/0.02% (w/v) of trypsin in EDTA solution (Biochrom, Germany) was added to the cell culture and they were incubated at 37°C and with 5% CO2 for 5 min. Trypsin activity was neutralized by adding medium and centrifugation at 150 g for 5 min.

    Scanning electron microscopy

    For the scanning electron microscopy (SEM) analysis, cells with a cell number of 6×104 were grown on 12 mm round glass coverslips for 24 h. They were fixed with modified 1% glutaraldehyde in 100 mM a phosphate buffer (DPBS, Gibco, Life Technologies, USA). After several washes in PBS, the samples were postfixed in 1% osmium tetroxide/PBS for 2 h on ice, washed in water, en bloc contrasted in 1% uranyl acetate/water, and dehydrated in a graded series of ethanol (30%, 50%, 70%, 90%, 96%, 2×100% ethanol; pure ethanol on molecular sieve). Finally, they were critical point dried using the Leica CPD 300 (Leica Microsystems, Vienna, Austria) and mounted on 12 mm aluminium stubs. Cells mounted on pieces of plastic were glued to conductive carbon pads and the edges of the plastic were surrounded with silver paint to reduce charging. Mounted samples were sputter coated with gold using the Baltec SCD 050 (Leica Microsystems, Vienna, Austria), and analyzed with a Jeol JSM 7500F cold field emission SEM (Jeol, Eching, Germany) at 8 mm working distance and 5 kV acceleration voltage using the lower secondary electron detector.

    SEM image analysis has been performed in ImageJ (NIH). 78 SEM images were obtained and manually screened for MNT bulges in total. Four parameters were determined for further analysis of the MNT ultrastructure: the MNT diameter, DMNT, the diameter of the bulge, DB, the distance between adjacent bulges, dBB, and the length of the bulges LB. LB was determined by fitting an oval to the MNT bulge and determining the length from one end of the oval to the other.

    Statistical analysis was conducted with JASP (https://jasp-stats.org). For comparison of distributions, the Bayesian independent samples t-test with a Cauchy scale of 0.5 was used. A Bayes factor of >10 was assigned as a threshold for a statistically significant difference of the distributions. Figure 1 was created using JASP and Adobe Illustrator.

    Acknowledgementslink

    The author would like to thank Dr. Thomas Kurth (TU Dresden, Center for Regenerative Therapies Dresden) for realization of electron microscopy and Simon Schaefer for proofreading the manuscript.

    Conflict of interestlink

    The authors declare no conflicts of interest.

    Ethics Statementlink

    Not applicable.

    No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by ScienceMatters.

    Referenceslink
    1. Gerdes, Hans-Hermann, Bukoreshtliev, Nickolay V., Barroso, João F. V.
      Tunneling nanotubes: A new route for the exchange of components between animal cells
      FEBS Letters, 581/2007, pages 2194-2201 chrome_reader_mode
    2. Hans-Hermann Gerdes, Rainer Pepperkok
      Cell-to-cell communication: current views and future perspectives
      Cell and Tissue Research, 352/2013, pages 1-3 chrome_reader_mode
    3. Ariazi, Jennifer, Benowitz, Andrew, de Biasi, Vern,more_horiz, Zurzolo, Chiara
      Tunneling Nanotubes and Gap Junctions–Their Role in Long-Range Intercellular Communication during Development, Health, and Disease Conditions
      Frontiers in Molecular Neuroscience, 10/2017, page 333 chrome_reader_mode
    4. Hans-Hermann Gerdes, Raquel Negrão Carvalho
      Intercellular transfer mediated by tunneling nanotubes
      Current Opinion in Cell Biology, 20/2008, pages 470-475 chrome_reader_mode
    5. Marzo, Ludovica, Gousset, Karine, Zurzolo, Chiara
      Multifaceted Roles of Tunneling Nanotubes in Intercellular Communication
      Frontiers in Physiology, 3/2012, page 72 chrome_reader_mode
    6. Wittig, Dierk, Wang, Xiang, Walter, Cindy,more_horiz, Roehlecke, Cora
      Multi-Level Communication of Human Retinal Pigment Epithelial Cells via Tunneling Nanotubes
      PLOS ONE, 7/2012, page e33195 chrome_reader_mode
    7. Zhang, Jianghui, Zhang, Youyi
      Membrane nanotubes: Novel communication between distant cells
      Science China Life Sciences, 56/2013, pages 994-999 chrome_reader_mode
    8. McCoy-Simandle, Kessler, Hanna, Samer J., Cox, Dianne
      Exosomes and nanotubes: Control of immune cell communication
      The International Journal of Biochemistry & Cell Biology, 71/2016, pages 44-54 chrome_reader_mode
    9. Austefjord, Magnus Wiger, Gerdes, Hans-Hermann, Wang, Xiang
      Tunneling nanotubes
      Communicative & Integrative Biology, 7/2014, page e27934 chrome_reader_mode
    10. Spafford, J. David, Wang, Xiang, Bukoreshtliev, Nickolay Vassilev, Gerdes, Hans-Hermann
      Developing Neurons Form Transient Nanotubes Facilitating Electrical Coupling and Calcium Signaling with Distant Astrocytes
      PLOS ONE, 7/2012, page e47429 chrome_reader_mode
    11. Amin Rustom
      The missing link: does tunnelling nanotube-based supercellularity provide a new understanding of chronic and lifestyle diseases?
      Open Biology, 6/2016, page 160057 chrome_reader_mode
    12. Felix Scholkmann
      Long range physical cell-to-cell signalling via mitochondria inside membrane nanotubes: a hypothesis
      Theoretical Biology and Medical Modelling, 13/2016, page 16 chrome_reader_mode
    13. Felix Scholkmann
      Two emerging topics regarding long-range physical signaling in neurosystems: Membrane nanotubes and electromagnetic fields
      Journal of Integrative Neuroscience, 14/2015, pages 135-153 chrome_reader_mode
    14. Björn Önfelt, Shlomo Nedvetzki, Kumiko Yanagi, Daniel M. Davis
      Cutting Edge: Membrane Nanotubes Connect Immune Cells
      The Journal of Immunology, 173/2004, pages 1511-1513 chrome_reader_mode
    15. Holly R. Chinnery, Eric Pearlman, Paul G. McMenamin
      Cutting Edge: Membrane Nanotubes In Vivo: A Feature of MHC Class II+ Cells in the Mouse Cornea
      The Journal of Immunology, 180/2008, pages 5779-5783 chrome_reader_mode
    16. X Sun, Y Wang, J Zhang, J Tu, X-J Wang, X-D Su, L Wang, Y Zhang
      Tunneling-nanotube direction determination in neurons and astrocytes
      Cell Death & Disease, 3/2012, page e438 chrome_reader_mode
    17. Krishna C. Vallabhaneni, Hermann Haller, Inna Dumler
      Vascular Smooth Muscle Cells Initiate Proliferation of Mesenchymal Stem Cells by Mitochondrial Transfer via Tunneling Nanotubes
      Stem Cells and Development, 21/2012, pages 3104-3113 chrome_reader_mode
    18. Kaiming Liu, Kunqian Ji, Liang Guo,more_horiz, Chuanzhu Yan
      Mesenchymal stem cells rescue injured endothelial cells in an in vitro ischemia–reperfusion model via tunneling nanotube like structure-mediated mitochondrial transfer
      Microvascular Research, 92/2014, pages 10-18 chrome_reader_mode
    19. X Wang, H-H Gerdes
      Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells
      Cell Death and Differentiation, 22/2015, pages 1181-1191 chrome_reader_mode
    20. Doreen Reichert, Julia Scheinpflug, Jana Karbanová, Daniel Freund, Martin Bornhäuser, Denis Corbeil
      Tunneling nanotubes mediate the transfer of stem cell marker CD133 between hematopoietic progenitor cells
      Experimental Hematology, 44/2016, pages 1092-1112.e2 chrome_reader_mode
    21. Ian Parker, Katrina T. Evans, Kyle Ellefsen, Devon A. Lawson, Ian F. Smith
      Lattice light sheet imaging of membrane nanotubes between human breast cancer cells in culture and in brain metastases
      Scientific Reports, 7/2017, page 11029 chrome_reader_mode
    22. Pooja Patheja, Khageswar Sahu
      Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments
      Experimental Cell Research, 355/2017, pages 182-193 chrome_reader_mode
    23. Bantseev, Vladimir, Youn, Hyun-Yi
      Mitochondrial “Movement” and Lens Optics following Oxidative Stress from UV-B Irradiation
      Annals of the New York Academy of Sciences, 1091/2006, pages 17-33 chrome_reader_mode
    24. Zanelli, Santina A., Trimmer, Patricia A., Solenski, Nina J.
      Nitric oxide impairs mitochondrial movement in cortical neurons during hypoxia
      Journal of Neurochemistry, 97/2006, pages 724-736 chrome_reader_mode
    25. Jennifer J. Smith, John D. Aitchison
      Peroxisomes take shape
      Nature Reviews Molecular Cell Biology, 14/2013, pages 803-817 chrome_reader_mode
    26. Kennady, P. Kavin, Ormerod, M. G., Singh, Shashi, Pande, Gopal
      Variation of mitochondrial size during the cell cycle: A multiparameter flow cytometric and microscopic study
      Cytometry Part A, 62A/2004, pages 97-108 chrome_reader_mode
    27. Timothy Wai, Thomas Langer
      Mitochondrial Dynamics and Metabolic Regulation
      Trends in Endocrinology & Metabolism, 27/2016, pages 105-117 chrome_reader_mode
    28. Acquistapace, Adrien, Bru, Thierry, Lesault, Pierre-François,more_horiz, Rodriguez, Anne-Marie
      Human Mesenchymal Stem Cells Reprogram Adult Cardiomyocytes Toward a Progenitor-Like State Through Partial Cell Fusion and Mitochondria Transfer
      STEM CELLS, 29/2011, pages 812-824 chrome_reader_mode
    29. Jennifer Pasquier, Bella S Guerrouahen, Hamda Al Thawadi, Pegah Ghiabi, Mahtab Maleki, Nadine Abu-Kaoud, Arthur Jacob, Massoud Mirshahi, Ludovic Galas, Shahin Rafii, Frank Le Foll, Arash Rafii
      Preferential transfer of mitochondria from endothelial to cancer cells through tunneling nanotubes modulates chemoresistance
      Journal of Translational Medicine, 11/2013, page 94 chrome_reader_mode
    30. Xiang Li, Yuelin Zhang, Sze C. Yeung,more_horiz, Qizhou Lian
      Mitochondrial Transfer of Induced Pluripotent Stem Cell–Derived Mesenchymal Stem Cells to Airway Epithelial Cells Attenuates Cigarette Smoke– Induced Damage
      American Journal of Respiratory Cell and Molecular Biology, 51/2014, pages 455-465 chrome_reader_mode
    31. Gregson, N. A., Williams, P. L.
      A Comparative Study of Brain and Liver Mitochondria from New-Born and Adult Rats
      Journal of Neurochemistry, 16/1969, pages 617-626 chrome_reader_mode
    32. Ilja Bobylev, Abhijeet R. Joshi, Mohammed Barham, Wolfram F. Neiss, Helmar C. Lehmann
      Depletion of Mitofusin-2 Causes Mitochondrial Damage in Cisplatin-Induced Neuropathy
      Molecular Neurobiology, 55/2018, pages 1227-1235 chrome_reader_mode
    33. Kolossov, Vladimir L., Sivaguru, Mayandi, Huff, Joseph,more_horiz, Gaskins, H. Rex
      Airyscan super-resolution microscopy of mitochondrial morphology and dynamics in living tumor cells
      Microscopy Research and Technique, 81/2018, pages 115-128 chrome_reader_mode
    34. Janos Feher, Illes Kovacs, Marco Artico, Carlo Cavallotti, Antonio Papale, Corrado Balacco Gabrieli
      Mitochondrial alterations of retinal pigment epithelium in age-related macular degeneration
      Neurobiology of Aging, 27/2006, pages 983-993 chrome_reader_mode
    35. Clotilde Théry, Laurence Zitvogel, Sebastian Amigorena
      Exosomes: composition, biogenesis and function
      Nature Reviews Immunology, 2/2002, pages 569-579 chrome_reader_mode
    36. Jin, Xin, Hwang, James C. M., Farina, Marco,more_horiz, Provinciali, Mauro
      Imaging of exosomes by broadband scanning microwave microscopy
      2016 46th European Microwave Conference (EuMC), 2016, pages 1211-1214 chrome_reader_mode
    37. Tavi, Pasi, Korhonen, Topi, Hänninen, Sandra L.,more_horiz, Westerblad, Håkan
      Myogenic skeletal muscle satellite cells communicate by tunnelling nanotubes
      Journal of Cellular Physiology, 223/2010, pages 376-383 chrome_reader_mode
    38. Amin Rustom, Rainer Saffrich, Ivanka Markovic, Paul Walther, Hans-Hermann Gerdes
      Nanotubular Highways for Intercellular Organelle Transport
      Science, 303/2004, pages 1007-1010 chrome_reader_mode
    39. Bénard, Magalie, Schapman, Damien, Lebon, Alexis,more_horiz, Galas, Ludovic
      Structural and functional analysis of tunneling nanotubes (TnTs) using gCW STED and gconfocal approaches
      Biology of the Cell, 107/2015, pages 419-425 chrome_reader_mode
    40. Minoo Razi, Sharon A. Tooze
      Chapter 17 Correlative Light and Electron Microscopy
      Autophagy in Mammalian Systems, Part B: Methods in Enzymology, 452/2009, pages 261-275 chrome_reader_mode
    41. Matthew R. G. Russell, Thomas R. Lerner, Jemima J. Burden, David O. Nkwe, Annegret Pelchen-Matthews, Marie-Charlotte Domart, Joanne Durgan, Anne Weston, Martin L. Jones, Christopher J. Peddie, Raffaella Carzaniga, Oliver Florey, Mark Marsh, Maximiliano G. Gutierrez, Lucy M. Collinson
      3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy
      Journal of Cell Science, 130/2017, pages 278-291 chrome_reader_mode
    Commentslink

    Create a Matters account to leave a comment.