Dataset Information

An Automated Image Analysis System to Quantify Endosomal Tubulation.

ABSTRACT: Recycling of cargos from early endosomes requires regulation of endosomal tubule formation and fission. This regulation is disrupted in cells depleted of the microtubule severing enzyme spastin, causing elongation of endosomal tubules and mis-trafficking of recycling endosomal cargos such as the transferrin receptor. Spastin is encoded by SPAST, mutations in which are the most frequent cause of autosomal dominant hereditary spastic paraplegia, a condition characterised by a progressive loss of lower limb function resulting from upper motor neuron axonopathy. Investigation of molecular factors involved in endosomal tubule regulation is hindered by the need for manual counting of endosomal tubules. We report here the development of an open source automated system for the quantification of endosomal tubules, using ImageJ and R. We validate the method in cells depleted of spastin and its binding partner IST1. The additional speed and reproducibility of this system compared with manual counting makes feasible screens of candidates to further understand the mechanisms of endosomal tubule formation and fission.

SUBMITTER: Newton TM

PROVIDER: S-EPMC5179261 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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An Automated Image Analysis System to Quantify Endosomal Tubulation.

Newton Timothy M TM Reid Evan E

PloS one 20161222 12

Recycling of cargos from early endosomes requires regulation of endosomal tubule formation and fission. This regulation is disrupted in cells depleted of the microtubule severing enzyme spastin, causing elongation of endosomal tubules and mis-trafficking of recycling endosomal cargos such as the transferrin receptor. Spastin is encoded by SPAST, mutations in which are the most frequent cause of autosomal dominant hereditary spastic paraplegia, a condition characterised by a progressive loss of l ...[more]

PMID: 28006827

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Project description:In response to injury, adult Schwann cells (SCs) re-enter the cell cycle, change their expression profile, and exert repair functions important for wound healing and the re-growth of axons. While this phenotypical instability of SCs is essential for nerve regeneration, it has also been implicated in cancer progression and de-myelinating neuropathies. Thus, SCs became an important research tool to study the molecular mechanisms involved in repair and disease and to identify targets for therapeutic intervention. A high purity of isolated SC cultures used for experimentation must be demonstrated to exclude that novel findings are derived from a contaminating fibroblasts population. In addition, information about the SC proliferation status is an important parameter to be determined in response to different treatments. The evaluation of SC purity and proliferation, however, usually depends on the time consuming, manual assessment of immunofluorescence stainings or comes with the sacrifice of a large amount of SCs for flow cytometry analysis. We here show that rat SC culture derived cytospins stained for SC marker SOX10, proliferation marker EdU, intermediate filament vimentin and DAPI allowed the determination of SC identity and proliferation by requiring only a small number of cells. Furthermore, the CellProfiler software was used to develop an automated image analysis pipeline that quantified SCs and proliferating SCs from the obtained immunofluorescence images. By comparing the results of total cell count, SC purity and SC proliferation rate between manual counting and the CellProfiler output, we demonstrated applicability and reliability of the established pipeline. In conclusion, we here combined the cytospin technique, a multi-colour immunofluorescence staining panel, and an automated image analysis pipeline to enable the quantification of SC purity and SC proliferation from small cell aliquots. This procedure represents a solid read-out to simplify and standardize the quantification of primary SC culture purity and proliferation.

Dataset Information

An Automated Image Analysis System to Quantify Endosomal Tubulation.

Publications

An Automated Image Analysis System to Quantify Endosomal Tubulation.

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