Confocal micrographs: automated segmentation and quantitative shape analysis of neuronal cells treated with ostreolysin A/pleurotolysin B pore-forming complex

Kopanja, Lazar; Kovacevic, Zorana; Tadic, Marin; Žužek, Monika Cecilija; Vrecl, Milka; Frangež, Robert

doi:10.1007/s00418-018-1670-0

Short Communication
Published: 23 April 2018

Confocal micrographs: automated segmentation and quantitative shape analysis of neuronal cells treated with ostreolysin A/pleurotolysin B pore-forming complex

Lazar Kopanja^1,2,
Zorana Kovacevic³,
Marin Tadic⁴,
Monika Cecilija Žužek⁵,
Milka Vrecl⁵ &
Robert Frangež⁵

Histochemistry and Cell Biology volume 150, pages 93–102 (2018)Cite this article

391 Accesses
5 Citations
4 Altmetric
Metrics details

Abstract

Detailed shape analysis of cells is important to better understand the physiological mechanisms of toxins and determine their effects on cell morphology. This study aimed to develop a procedure for accurate morphological analysis of cell shape and use it as a tool to estimate toxin activity. With the aim of optimizing the method of cell morphology analysis, we determined the influence of ostreolysin A and pleurotolysin B complex (OlyA/PlyB) on the morphology of murine neuronal NG108-15 cells. A computational method was introduced and successfully applied to quantify morphological attributes of the NG108-15 cell line before and after 30 and 60 min exposure to OlyA/PlyB using confocal microscopy. The modified circularity measure \(C_{2}^{n}(S)\) for shape analysis was applied, which defines the degree to which the shape of the neuron differs from a perfect circle. It enables better detection of small changes in the shape of cells, making the outcome easily detectable numerically. Additionally, we analyzed the influence of OlyA/PlyB on the cell area, allowing us to detect the cells with blebs. This is important because the formation of plasma membrane protrusions such as blebs often reflects cell injury that leads to necrotic cell death. In summary, we offer a novel analytical method of neuronal cell shape analysis and its correlation with the toxic effects of the pore-forming OlyA/PlyB toxin in situ.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

References

Al-Deen IHS, Twaij HAA, Al-Badr AA, Istarabadi TAW (1987) Toxicologic and histopathologic studies of Pleurotus ostreatus mushroom in mice. J Ethnopharmacol 21:297–305. https://doi.org/10.1016/0378-8741(87)90105-X
Article PubMed CAS Google Scholar
Al-Fahdawi S, Qahwaji R, Al-Waisy AS, Ipson S, Malik RA, Brahma A, Chen X (2016) A fully automatic nerve segmentation and morphometric parameter quantification system for early diagnosis of diabetic neuropathy in corneal images. Comput Methods Programs Biomed 135:151–166. https://doi.org/10.1016/j.cmpb.2016.07.032
Article PubMed Google Scholar
Apopa PL, He X, Ma Q (2008) Phosphorylation of Nrf2 in the transcription activation domain by casein kinase 2 (CK2) is critical for the nuclear translocation and transcription activation function of Nrf2 in IMR-32 neuroblastoma cells. J Biochem Mol Toxicol 22:63–76. https://doi.org/10.1002/jbt.20212
Article PubMed CAS Google Scholar
Arciello M, Rotilio G, Rossi L (2005) Copper-dependent toxicity in SH-SY5Y neuroblastoma cells involves mitochondrial damage. Biochem Biophys Res Commun 327:454–459. https://doi.org/10.1016/j.bbrc.2004.12.022
Article PubMed CAS Google Scholar
Auman HJ, Coleman H, Riley HE, Olale F, Tsai HJ, Yelon D (2007) Functional modulation of cardiac form through regionally confined cell shape changes. PLoS Biol 5:e53. https://doi.org/10.1371/journal.pbio.0050053
Article PubMed PubMed Central CAS Google Scholar
Babiychuk E, Monastyrskaya K, Potez S, Draeger A (2011) Blebbing confers resistance against cell lysis. Cell Death Differ 18:80–89. https://doi.org/10.1038/cdd.2010.81
Article PubMed CAS Google Scholar
Bastaki SMA, Adeghate E, Chandranath IS, Amir N, Tariq S, Hameed RS, Adem A (2010) Effects of streptozotocin-induced long-term diabetes on parietal cell function and morphology in rats. Mol Cell Biochem 341:43–50. https://doi.org/10.1007/s11010-010-0435-4
Article PubMed CAS Google Scholar
Bora K, Chowdhury M, Mahanta LB, Kundu MK, Das AK (2017) Automated classification of Pap smear images to detect cervical dysplasia. Comput Methods Programs Biomed 138:31–47. https://doi.org/10.1016/j.cmpb.2016.10.001
Article PubMed Google Scholar
Castoldi AF, Coccini T, Ceccatelli S, Manzo L (2001) Neurotoxicity and molecular effects of methylmercury. Brain Res Bull 55:197–203. https://doi.org/10.1016/S0361-9230(01)00458-0
Article PubMed CAS Google Scholar
Chen KC, Nicholson C (2000) Changes in brain cell shape create residual extracellular space volume and explain tortuosity behavior during osmotic challenge. Proc Natl Acad Sci 97:8306–8311. https://doi.org/10.1073/pnas.150338197
Article PubMed CAS Google Scholar
Chen YC, Chen YC, Chiang AS (2008) Template-driven segmentation of confocal microscopy images. Comput Methods Programs Biomed 89:239–247. https://doi.org/10.1016/j.cmpb.2007.11.007
Article PubMed Google Scholar
Chen X, Chen K, He J, Yao K (2016) Comparing the curative cffects between femtosecond laser-assisted cataract surgery and conventional phacoemulsification surgery: a meta-analysis. PLoS One 11:e0152088. https://doi.org/10.1371/journal.pone.0152088
Article PubMed PubMed Central CAS Google Scholar
Delgado-González JC, Mansilla-Legorburo F, Florensa-Vila J, Insausti AM, Viñuela A, Tuñón-Alvarez T, Cruz M, Mohedano-Moriano A, Insausti R, Artacho-Pérula E (2015) Quantitative measurements in the human hippocampus and related areas: correspondence between ex-vivo MRI and histological preparations. PLoS One 10:e0130314. https://doi.org/10.1371/journal.pone.0130314
Article PubMed PubMed Central CAS Google Scholar
Donkin JJ, Vink R (2010) Mechanisms of cerebral edema in traumatic brain injury: therapeutic developments. Curr Opin Neurol 23:293–299. https://doi.org/10.1097/WCO.0b013e328337f451
Article PubMed CAS Google Scholar
Flamier A, Singh S, Rasmussen TP (2017) A standardized human embryoid body platform for the detection and analysis of teratogens. PLoS One 12:e0171101. https://doi.org/10.1371/journal.pone.0171101
Article PubMed PubMed Central CAS Google Scholar
Gelzinis A, Verikas A, Vaiciukynas E, Bacauskiene M, Sulcius S, Simoliunas E, Staniulis J, Paskauskas R (2015) Automatic detection and morphological delineation of bacteriophages in electron microscopy images. Comput Biol Med 64:101–116. https://doi.org/10.1016/j.compbiomed.2015.06.015
Article PubMed CAS Google Scholar
Gould R, Bassen DM, Chakrabarti A, Varner JD, Butcher J (2016) Population heterogeneity in the epithelial to mesenchymal transition is controlled by NFAT and phosphorylated Sp1. PLOS Comput Biol 12:e1005251. https://doi.org/10.1371/journal.pcbi.1005251
Article PubMed PubMed Central CAS Google Scholar
Han RI, Wheeler TM, Lumsden AB, Reardon MJ, Lawrie GM, Grande-Allen KJ, Morrisett JD, Brunner G (2016) Morphometric analysis of calcification and fibrous layer thickness in carotid endarterectomy tissues. Comput Biol Med 70:210–219. https://doi.org/10.1016/j.compbiomed.2016.01.019
Article PubMed PubMed Central Google Scholar
Huet C, Pisselet C, Mandon-Pépin B, Monget P, Monniaux D (2001) Extracellular matrix regulates ovine granulosa cell survival, proliferation and steroidogenesis: relationships between cell shape and function. J Endocrinol 169:347–360. https://doi.org/10.1677/JOE.0.1690347
Article PubMed CAS Google Scholar
Keller HU, Niggli V, Zimmermann A (1989) Diacylglycerols and PMA induce actin polymerization and distinct shape changes in lymphocytes: relation to fluid pinocytosis and locomotion. J Cell Sci 93(Pt 3):457–465
PubMed CAS Google Scholar
Kopanja L, Kralj S, Zunic D, Loncar B, Tadic M (2016a) Core–shell superparamagnetic iron oxide nanoparticle (SPION) clusters: TEM micrograph analysis, particle design and shape analysis. Ceram Int 42:10976–10984. https://doi.org/10.1016/j.ceramint.2016.03.235
Article CAS Google Scholar
Kopanja L, Milosevic I, Panjan M, Damnjanovic V, Tadic M (2016b) Sol–gel combustion synthesis, particle shape analysis and magnetic properties of hematite (α-Fe₂O₃) nanoparticles embedded in an amorphous silica matrix. Appl Surf Sci 362:380–386. https://doi.org/10.1016/j.apsusc.2015.11.238
Article CAS Google Scholar
Kotyk T, Dey N, Ashour AS, Balas-Timar D, Chakraborty S, Ashour AS, Tavares JM (2016) Measurement of glomerulus diameter and Bowman’s space width of renal albino rats. Comput Methods Programs Biomed 126:143–153. https://doi.org/10.1016/j.cmpb.2015.10.023
Article PubMed Google Scholar
Lai-Fook SJ, Beck KC, Sutcliffe AM, Donaldson JT (1984) Effect of edema and height on bronchial diameter and shape in excised dog lung. Respir Physiol 55:223–237. https://doi.org/10.1016/0034-5687(84)90024-0
Article PubMed CAS Google Scholar
Michaels JA, Dann B, Scherberger H (2016) Neural population dynamics during reaching are better explained by a dynamical system than representational tuning. PLOS Comput Biol 12:e1005175. https://doi.org/10.1371/journal.pcbi.1005175
Article PubMed PubMed Central CAS Google Scholar
Ming-Kuei Hu (1962) Visual pattern recognition by moment invariants. IRE Trans Inf Theory 8:179–187. https://doi.org/10.1109/TIT.1962.1057692
Article Google Scholar
Muscas G, Singh G, Glomm WR, Mathieu R, Kumar PA, Concas G, Agostinelli E, Peddis D (2015) Tuning the size and shape of oxide nanoparticles by controlling oxygen content in the reaction environment: morphological analysis by aspect maps. Chem Mater 27:1982–1990. https://doi.org/10.1021/cm5038815
Article CAS Google Scholar
Ota K, Leonardi A, Mikelj M, Skočaj M, Wohlschlager T, Künzler M, Aebi M, Narat M, Križaj I, Anderluh G, Sepčić K, Maček P (2013) Membrane cholesterol and sphingomyelin, and ostreolysin A are obligatory for pore-formation by a MACPF/CDC-like pore-forming protein, pleurotolysin B. Biochimie 95:1855–1864. https://doi.org/10.1016/j.biochi.2013.06.012
Article PubMed CAS Google Scholar
Ota K, Butala M, Viero G, Dalla Serra M, Sepčić K, Maček P (2014) Fungal MACPF-like proteins and aegerolysins: bi-component pore-forming proteins? Subcell Biochem 80:271–291. https://doi.org/10.1007/978-94-017-8881-6_14
Article PubMed CAS Google Scholar
Otsu N (1979) A threshold selection method from gray-level histograms. IEEE Trans Syst Man Cybern 9:62–66. https://doi.org/10.1109/TSMC.1979.4310076
Article Google Scholar
Pollard TD, Cooper JA (2009) Actin, a central player in cell shape and movement. Science 326:1208–1212. https://doi.org/10.1126/science.1175862
Article PubMed PubMed Central CAS Google Scholar
Rungta RL, Choi HB, Tyson JR, Malik A, Dissing-Olesen L, Lin PJC, Cain SM, Cullis PR, Snutch TP, MacVicar BA (2015) The cellular mechanisms of neuronal swelling underlying cytotoxic edema. Cell 161:610–621. https://doi.org/10.1016/j.cell.2015.03.029
Article PubMed CAS Google Scholar
Sosa JM, Huber DE, Welk B, Fraser HL (2014) Development and application of MIPAR™: a novel software package for two- and three-dimensional microstructural characterization. Integr Mater Manuf Innov 3:10. https://doi.org/10.1186/2193-9772-3-10
Article Google Scholar
Trump BE, Berezesky IK, Seung H, Phelps PC (1997) The pathways of cell death: oncosis, apoptosis, and necrosis. Toxicol Pathol 25:82–88. https://doi.org/10.1177/019262339702500116
Article PubMed CAS Google Scholar
Tsai WB, Lin JH (2009) Modulation of morphology and functions of human hepatoblastoma cells by nano-grooved substrata. Acta Biomater 5:1442–1454. https://doi.org/10.1016/j.actbio.2009.01.002
Article PubMed CAS Google Scholar
Van Beers EJ, Samsel L, Mendelsohn L, Saiyed R, Fertrin KY, Brantner CA, Daniels MP, Nichols J, McCoy JP, Kato GJ (2014) Imaging flow cytometry for automated detection of hypoxia-induced erythrocyte shape change in sickle cell disease. Am J Hematol 89:598–603. https://doi.org/10.1002/ajh.23699
Article PubMed PubMed Central CAS Google Scholar
Vincent L, Soille P (1991) Watersheds in digital spaces: an efficient algorithm based on immersion simulations. IEEE Trans Pattern Anal Mach Intell 13:583–598. https://doi.org/10.1109/34.87344
Article Google Scholar
Vrecl M, Babnik M, Diacci U, Benoit E, Frangež R (2015a) Effect of the Ostreolysin A/Pleurotolysin B pore-forming complex on neuroblastoma cell morphology and intracellular Ca²⁺ activity. Toxicol Sci 144:276–283. https://doi.org/10.1093/toxsci/kfu316
Article PubMed CAS Google Scholar
Vrecl M, Babnik M, Sepčić K, Žužek MC, Maček P, Diacci U, Frangež R (2015b) Effect of the ostreolysin A/pleurotolysin B pore-forming complex on intracellular Ca²⁺ activity in the vascular smooth muscle cell line A10. Toxicol In Vitro 29:2015–2021. https://doi.org/10.1016/j.tiv.2015.08.014
Article PubMed CAS Google Scholar
White JG, Amos WB, Fordham M (1987) An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J Cell Biol 105:41–48. https://doi.org/10.1083/jcb.105.1.41
Article PubMed CAS Google Scholar
Žunić J, Hirota K (2008) Measuring Shape Circularity. In: Ruiz-Shulcloper J, Kropatsch WG (eds) Progress in pattern recognition, image analysis and applications. CIARP 2008. Lecture Notes in Computer Science, vol 5197. Springer, Berlin, pp 94–101. https://doi.org/10.1007/978-3-540-85920-8_12
Chapter Google Scholar
Žunić J, Hirota K, Rosin PL (2010) A Hu moment invariant as a shape circularity measure. Pattern Recognit 43:47–57. https://doi.org/10.1016/j.patcog.2009.06.017
Article Google Scholar
Žunić J, Hirota K, Rosin PL (2014) Note on the shape circularity measure method based on radial moments. J Electron Imaging 23:029701. https://doi.org/10.1117/1.JEI.23.2.029701
Article Google Scholar
Žužek MC, Maček P, Sepčić K, Cestnik V, Frangež R (2006) Toxic and lethal effects of ostreolysin, a cytolytic protein from edible oyster mushroom (Pleurotus ostreatus), in rodents. Toxicon 48:264–271. https://doi.org/10.1016/j.toxicon.2006.05.011
Article PubMed CAS Google Scholar

Download references

Acknowledgements

This work was supported by the Serbian Ministry of Education, Science and Technological Development and the Slovenian Research Agency program P4-0053.

Author information

Affiliations

Faculty of Mathematics and Computer Science, Alfa BK University, Belgrade, Serbia
Lazar Kopanja
Faculty of Technology and Metallurgy, University of Belgrade, Belgrade, Serbia
Lazar Kopanja
Department of Veterinary Medicine, Faculty of Agriculture, University of Novi Sad, Novi Sad, Serbia
Zorana Kovacevic
Condensed Matter Physics Laboratory, Vinca Institute, University of Belgrade, Belgrade, Serbia
Marin Tadic
Institute of Preclinical Sciences, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia
Monika Cecilija Žužek, Milka Vrecl & Robert Frangež

Authors

Lazar Kopanja
View author publications
You can also search for this author in PubMed Google Scholar
Zorana Kovacevic
View author publications
You can also search for this author in PubMed Google Scholar
Marin Tadic
View author publications
You can also search for this author in PubMed Google Scholar
Monika Cecilija Žužek
View author publications
You can also search for this author in PubMed Google Scholar
Milka Vrecl
View author publications
You can also search for this author in PubMed Google Scholar
Robert Frangež
View author publications
You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Robert Frangež.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kopanja, L., Kovacevic, Z., Tadic, M. et al. Confocal micrographs: automated segmentation and quantitative shape analysis of neuronal cells treated with ostreolysin A/pleurotolysin B pore-forming complex. Histochem Cell Biol 150, 93–102 (2018). https://doi.org/10.1007/s00418-018-1670-0

Download citation

Accepted: 17 April 2018
Published: 23 April 2018
Issue Date: July 2018
DOI: https://doi.org/10.1007/s00418-018-1670-0

Keywords

Image analyses
Confocal microscopy
Cell morphology
Cell shape
Ostreolysin A/pleurotolysin B