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Protocol and Software for Automated Detection of Lysosome Active “Runs” and “Flights” with Wavelet Transform Approach

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Signal Transduction Immunohistochemistry

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2593))

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Abstract

Lysosomes are highly dynamic degradation/recycling organelles that harbor sophisticated molecular sensors and signal transduction machinery through which they control cell adaptation to environmental cues and nutrients. The movements of these signaling hubs comprise persistent, directional runs—active, ATP-dependent transport along the microtubule tracks—interspersed by short, passive movements and pauses imposed by cytoplasmic constraints. The trajectories of individual lysosomes are usually obtained by time-lapse imaging of the acidic organelles labeled with LysoTracker dyes or fluorescently-tagged lysosomal-associated membrane proteins LAMP1 and LAMP2. Subsequent particle tracking generates large data sets comprising thousands of lysosome trajectories and hundreds of thousands of data points. Analyzing such data sets requires unbiased, automated methods to handle large data sets while capturing the temporal heterogeneity of lysosome trajectory data. This chapter describes integrated and largely automated workflow from live cell imaging to lysosome trajectories to computing the parameters of lysosome dynamics. We describe an open-source code for implementing the continuous wavelet transform (CWT) to distinguish trajectory segments corresponding to active transport (i.e., “runs” and “flights”) versus passive lysosome movements. Complementary cumulative distribution functions (CDFs) of the “runs/flights” are generated, and Akaike weight comparisons with several competing models (lognormal, power law, truncated power law, stretched exponential, exponential) are performed automatically. Such high-throughput analyses yield useful aggregate/ensemble metrics for lysosome active transport.

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References

  1. Holland LKK, Nielsen IO, Maeda K et al (2020) Snapshot: lysosomal functions. Cell 181:748–748 e741

    Article  CAS  Google Scholar 

  2. Saftig P, Klumperman J (2009) Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat Rev Mol Cell Biol 10:623–635

    Article  CAS  Google Scholar 

  3. Ballabio A, Bonifacino JS (2020) Lysosomes as dynamic regulators of cell and organismal homeostasis. Nat Rev Mol Cell Biol 21:101–118

    Article  CAS  Google Scholar 

  4. Lawrence RE, Zoncu R (2019) The lysosome as a cellular Centre for signalling, metabolism and quality control. Nat Cell Biol 21:133–142

    Article  CAS  Google Scholar 

  5. Savini M, Zhao Q, Wang MC (2019) Lysosomes: Signaling hubs for metabolic sensing and longevity. Trends Cell Biol 29:876–887

    Article  CAS  Google Scholar 

  6. Inpanathan S, Botelho RJ (2019) The lysosome signaling platform: adapting with the times. Front Cell Dev Biol 7:113

    Article  Google Scholar 

  7. Cao M, Luo X, Wu K et al (2021) Targeting lysosomes in human disease: from basic research to clinical applications. Signal Transduct Target Ther 6:379

    Article  CAS  Google Scholar 

  8. Korolchuk VI, Saiki S, Lichtenberg M et al (2011) Lysosomal positioning coordinates cellular nutrient responses. Nat Cell Biol 13:453–460

    Article  CAS  Google Scholar 

  9. Cordonnier MN, Dauzonne D, Louvard D et al (2001) Actin filaments and myosin I alpha cooperate with microtubules for the movement of lysosomes. Mol Biol Cell 12:4013–4029

    Article  CAS  Google Scholar 

  10. Balabanian L, Lessard DV, Yaninska P et al (2021) Tau differentially regulates the transport of early endosomes and lysosomes. bioRxiv:2021.2011.2001.466759

    Google Scholar 

  11. Balint S, Verdeny Vilanova I, Sandoval Alvarez A et al (2013) Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections. Proc Natl Acad Sci U S A 110:3375–3380

    Article  CAS  Google Scholar 

  12. Ba Q, Raghavan G, Kiselyov K et al (2018) Whole-cell scale dynamic organization of lysosomes revealed by spatial statistical analysis. Cell Rep 23:3591–3606

    Article  CAS  Google Scholar 

  13. Matteoni R, Kreis TE (1987) Translocation and clustering of endosomes and lysosomes depends on microtubules. J Cell Biol 105:1253–1265

    Article  CAS  Google Scholar 

  14. Wang B, Kuo J, Granick S (2013) Bursts of active transport in living cells. Phys Rev Lett 111:208102

    Article  Google Scholar 

  15. Verdeny-Vilanova I, Wehnekamp F, Mohan N et al (2017) 3D motion of vesicles along microtubules helps them to circumvent obstacles in cells. J Cell Sci 130:1904–1916

    CAS  Google Scholar 

  16. Cabukusta B, Neefjes J (2018) Mechanisms of lysosomal positioning and movement. Traffic 19:761–769

    Article  CAS  Google Scholar 

  17. Pu J, Guardia CM, Keren-Kaplan T et al (2016) Mechanisms and functions of lysosome positioning. J Cell Sci 129:4329–4339

    CAS  Google Scholar 

  18. Harada A, Takei Y, Kanai Y et al (1998) Golgi vesiculation and lysosome dispersion in cells lacking cytoplasmic dynein. J Cell Biol 141:51–59

    Article  CAS  Google Scholar 

  19. Jordens I, Fernandez-Borja M, Marsman M et al (2001) The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors. Curr Biol 11:1680–1685

    Article  CAS  Google Scholar 

  20. Hollenbeck PJ, Swanson JA (1990) Radial extension of macrophage tubular lysosomes supported by kinesin. Nature 346:864–866

    Article  CAS  Google Scholar 

  21. Pu J, Schindler C, Jia R et al (2015) BORC, a multisubunit complex that regulates lysosome positioning. Dev Cell 33:176–188

    Article  CAS  Google Scholar 

  22. Koslover EF, Chan CK, Theriot JA (2016) Disentangling random motion and flow in a complex medium. Biophys J 110:700–709

    Article  CAS  Google Scholar 

  23. Polev K, Kolygina DV, Kandere-Grzybowska K et al (2022) Large-scale, wavelet-based analysis of lysosomal trajectories and co-movements of lysosomes with nanoparticle cargos. Cells 11:270

    Google Scholar 

  24. Smal I, Meijering E, Draegestein K et al (2008) Multiple object tracking in molecular bioimaging by Rao-Blackwellized marginal particle filtering. Med Image Anal 12:764–777

    Article  CAS  Google Scholar 

  25. Meijering E, Dzyubachyk O, Smal I (2012) Methods for cell and particle tracking. Methods Enzymol 504:183–200

    Article  Google Scholar 

  26. Jaqaman K, Danuser G (2009) Computational image analysis of cellular dynamics: a case study based on particle tracking. Cold Spring Harb Protoc 2009:pdb.top65

    Article  Google Scholar 

  27. Bandyopadhyay D, Cyphersmith A, Zapata JA et al (2014) Lysosome transport as a function of lysosome diameter. PLoS One 9:e86847

    Article  Google Scholar 

  28. Durso W, Martins M, Marchetti L et al (2020) Lysosome dynamic properties during neuronal stem cell differentiation studied by spatiotemporal fluctuation spectroscopy and organelle tracking. Int J Mol Sci 21:3397

    Article  Google Scholar 

  29. Zhao H, Zhou Q, Xia M et al (2018) Characterize collective lysosome heterogeneous dynamics in live cell with a space- and time-resolved method. Anal Chem 90:9138–9147

    Article  CAS  Google Scholar 

  30. Thompson GL, Beier HT, Ibey BL (2018) Tracking lysosome migration within Chinese hamster ovary (CHO) cells following exposure to nanosecond pulsed electric fields. Bioengineering (Basel) 5:103

    Article  CAS  Google Scholar 

  31. Mohan N, Sorokina EM, Verdeny IV et al (2019) Detyrosinated microtubules spatially constrain lysosomes facilitating lysosome-autophagosome fusion. J Cell Biol 218:632–643

    Article  CAS  Google Scholar 

  32. Han D, Korabel N, Chen R et al (2020) Deciphering anomalous heterogeneous intracellular transport with neural networks. elife 9:e52224

    Article  Google Scholar 

  33. Chen K, Wang B, Guan J et al (2013) Diagnosing heterogeneous dynamics in single-molecule/particle trajectories with multiscale wavelets. ACS Nano 7:8634–8344

    Article  CAS  Google Scholar 

  34. Chenouard N, Smal I, De Chaumont F et al (2014) Objective comparison of particle tracking methods. Nat Methods 11:281–289

    Article  CAS  Google Scholar 

  35. Jaqaman K, Loerke D, Mettlen M et al (2008) Robust single-particle tracking in live-cell time-lapse sequences. Nat Methods 5:695–702

    Article  CAS  Google Scholar 

  36. Ershov D, Phan M-S, Pylvänäinen JW et al (2021) Bringing TrackMate in the era of machine-learning and deep-learning. bioRxiv:2021.09.03.458852

    Google Scholar 

  37. Tinevez JY, Perry N, Schindelin J et al (2017) TrackMate: an open and extensible platform for single-particle tracking. Methods 115:80–90

    Article  CAS  Google Scholar 

  38. Clauset A, Shalizi CR, Newman MEJ (2009) Power-law distributions in empirical data. SIAM Rev 51:661–703

    Article  Google Scholar 

  39. Hofmann I, Munro S (2006) An N-terminally acetylated Arf-like GTPase is localised to lysosomes and affects their motility. J Cell Sci 119:1494–1503

    Article  CAS  Google Scholar 

  40. Arcizet D, Meier B, Sackmann E et al (2008) Temporal analysis of active and passive transport in living cells. Phys Rev Lett 101:248103

    Article  Google Scholar 

  41. Chen K, Wang B, Granick S (2015) Memoryless self-reinforcing directionality in endosomal active transport within living cells. Nat Mater 14:589–593

    Article  CAS  Google Scholar 

  42. Zaburdaev V, Denisov S, Klafter J (2015) Lévy walks. Rev Mod Phys 87:483–530

    Article  CAS  Google Scholar 

  43. Reynolds A (2015) Liberating Lévy walk research from the shackles of optimal foraging. Phys Life Rev 14:59–83

    Article  Google Scholar 

  44. Huda S, Weigelin B, Wolf K et al (2018) Lévy-like movement patterns of metastatic cancer cells revealed in microfabricated systems and implicated in vivo. Nat Commun 9:4539

    Article  Google Scholar 

  45. Fricke GM, Letendre KA, Moses ME et al (2016) Persistence and adaptation in immunity: T cells balance the extent and thoroughness of search. PLoS Comput Biol 12:e1004818

    Article  Google Scholar 

  46. Van Engelenburg SB, Palmer AE (2010) Imaging type-III secretion reveals dynamics and spatial segregation of Salmonella effectors. Nat Methods 7:325–330

    Article  Google Scholar 

  47. Borkowska M, Siek M, Kolygina DV et al (2020) Targeted crystallization of mixed-charge nanoparticles in lysosomes induces selective death of cancer cells. Nat Nanotechnol 15:331–341

    Article  CAS  Google Scholar 

  48. Kolygina DV, Siek M, Borkowska M et al (2021) Mixed-charge nanocarriers allow for selective targeting of mitochondria by otherwise nonselective dyes. ACS Nano 15:11470–11490

    Article  CAS  Google Scholar 

  49. De Jager M, Weissing FJ, Herman PM et al (2011) Levy walks evolve through interaction between movement and environmental complexity. Science 332:1551–1553

    Article  Google Scholar 

  50. Ghosh S, Dellibovi-Ragheb TA, Kerviel A et al (2020) Beta-coronaviruses use lysosomes for egress instead of the biosynthetic secretory pathway. Cell 183:1520–1535

    Article  CAS  Google Scholar 

  51. Farias GG, Guardia CM, De Pace R et al (2017) BORC/kinesin-1 ensemble drives polarized transport of lysosomes into the axon. Proc Natl Acad Sci U S A 114:E2955–E2964

    Article  CAS  Google Scholar 

  52. Humphries WHT, Szymanski CJ, Payne CK (2011) Endo-lysosomal vesicles positive for Rab7 and LAMP1 are terminal vesicles for the transport of dextran. PLoS One 6:e26626

    Article  CAS  Google Scholar 

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Correspondence to Kristiana Kandere-Grzybowska .

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Kandere-Grzybowska, K., Polev, K., Kolygina, D.V., Grzybowski, B.A. (2023). Protocol and Software for Automated Detection of Lysosome Active “Runs” and “Flights” with Wavelet Transform Approach. In: Kalyuzhny, A.E. (eds) Signal Transduction Immunohistochemistry. Methods in Molecular Biology, vol 2593. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2811-9_11

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  • DOI: https://doi.org/10.1007/978-1-0716-2811-9_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2810-2

  • Online ISBN: 978-1-0716-2811-9

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