Automated analysis of filopodial length and spatially resolved protein concentration via adaptive shape tracking

Saha, Tanumoy; Rathmann, Isabel; Viplav, Abhiyan; Panzade, Sadhana; Begemann, Isabell; Rasch, Coen R. N.; Klingauf, Jürgen; Matis, Maja; Galic, Milos

doi:10.1091/mbc.e16-06-0406

Cited by 13 publications

(12 citation statements)

References 37 publications

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“…To correlate the electrophysiological properties with the expression of neurochemical marker specific for different cortical interneuron types, we performed patch-clamp recordings with simultaneous filling of biocytin and the fluorescent dye Alexa 594 to allow an unambiguous identification of the recorded neuron. After determining the passive and active electrophysiological properties, slices were briefly fixed in PFA and processed for immunostaining for PV, SOM, and Prox1, a marker for vasoactive intestinal peptide-expressing interneurons ( Saha et al. 2016 ) and a fraction of reelin-and calretinin-expressing interneurons ( Rubin and Kessaris 2013 ; Miyoshi et al.…”

Section: Resultsmentioning

confidence: 99%

Layer-Specific Inhibitory Microcircuits of Layer 6 Interneurons in Rat Prefrontal Cortex

et al. 2020

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GABAergic interneurons in different cortical areas play important roles in diverse higher-order cognitive functions. The heterogeneity of interneurons is well characterized in different sensory cortices, in particular in primary somatosensory and visual cortex. However, the structural and functional properties of the medial prefrontal cortex (mPFC) interneurons have received less attention. In this study, a cluster analysis based on axonal projection patterns revealed four distinct clusters of L6 interneurons in rat mPFC: Cluster 1 interneurons showed axonal projections similar to Martinotti-like cells extending to layer 1, cluster 2 displayed translaminar projections mostly to layer 5, and cluster 3 interneuron axons were confined to the layer 6, whereas those of cluster 4 interneurons extend also into the white matter. Correlations were found between neuron location and axonal distribution in all clusters. Moreover, all cluster 1 L6 interneurons showed a monotonically adapting firing pattern with an initial high-frequency burst. All cluster 2 interneurons were fast-spiking, while neurons in cluster 3 and 4 showed heterogeneous firing patterns. Our data suggest that L6 interneurons that have distinct morphological and physiological characteristics are likely to innervate different targets in mPFC and thus play differential roles in the L6 microcircuitry and in mPFC-associated functions.

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Section: Resultsmentioning

confidence: 99%

Layer-Specific Inhibitory Microcircuits of Layer 6 Interneurons in Rat Prefrontal Cortex

et al. 2020

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“…FiloQuant is best suited for filopodia within 3D environments and does not incorporate protein fluorescence measurements ( Jacquemet et al, 2017 Preprint ). Saha et al (2016) have pioneered systematic fluorescence measurement within filopodia, looking at both tip fluorescence and fluorescence along the length of the filopodium, in low throughput. Filopodyan measures the fluorescence at the membrane-localized sites of actin incorporation at both tips and bases and links this with the parameters of filopodia morphology and dynamics in large datasets, in open source format and with a user-friendly graphical user interface.…”

Section: Discussionmentioning

confidence: 99%

“…Software has also been specifically designed for the automated detection of filopodia in dendrites, with a focus on their longitudinal and lateral movement ( Hendricusdottir and Bergmann, 2014 ; Tárnok et al, 2015 ). Saha et al (2016) pioneered concurrent analysis of fluorescence and filopodium behavior, developing MATLAB software for semiautomated analysis of filopodia dynamics and fluorescence. Most recently, the ImageJ plugin FiloQuant has enabled customizable automated quantification of filopodia lengths and densities, particularly in 3D microenvironments ( Jacquemet et al, 2017 Preprint ).…”

Section: Introductionmentioning

confidence: 99%

Filopodyan: An open-source pipeline for the analysis of filopodia

Urbančič

Butler

Richier

et al. 2017

Journal of Cell Biology

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“…The objective of these studies was to analyze dynamic changes in length, as well as vertical and lateral tilt of filopodia. These, as well as an additional technique designed to determine the presence of proteins along the length of the filopodium (Saha et al, ), are not suitable for data sets comprising thousands of filopodia. These methods were not congruent with the objective of the current studies, which was to understand prevalence in order to identify mechanisms for filopodia regulation.…”

Section: Methodsmentioning

confidence: 99%

The tumor promoter‐activated protein kinase Cs are a system for regulating filopodia

et al. 2017

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Different protein kinase C (PKC) isoforms have distinct roles in regulating cell functions. The conventional (α, β, γ) and novel (δ, ɛ, η, θ) classes are targets of phorbol ester tumor promoters, which are surrogates of endogenous second messenger, diacylglycerol. The promoter‐stimulated disappearance of filopodia was investigated by use of blocking peptides (BPs) that inhibit PKC maturation and/or docking. Filopodia were partially rescued by a peptide representing PKC ɛ hydrophobic sequence, but also by a myristoylated PKC α/β pseudosubstrate sequence, and an inhibitor of T‐cell protein tyrosine phosphatase (TC‐PTP). The ability to turn over filopodia was widely distributed among PKC isoforms. PKC α and η hydrophobic sequences enhanced filopodia in cells in the absence of tumor promoter treatment. With transcriptional knockdown of PKC α, the content of PKC ɛ predominated over other isoforms. PKC ɛ could decrease filopodia significantly in promoter‐treated cells, and this was attributed to ruffling. The presence of PKC α counteracted the PKC ɛ‐mediated enhancement of ruffling. The results showed that there were two mechanisms of filopodia downregulation. One operated in the steady‐state and relied on PKC α and η. The other was stimulated by tumor promoters and relied on PKC ɛ. Cycles of protrusion and retraction are characteristic of filopodia and are essential for the cell to orient itself during chemotaxis and haptotaxis. By suppressing filopodia, PKC ɛ can create a long‐term “memory” of an environmental signal that may act in nature as a mnemonic device to mark the direction of a repulsive signal.

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Automated analysis of filopodial length and spatially resolved protein concentration via adaptive shape tracking

Cited by 13 publications

References 37 publications

Layer-Specific Inhibitory Microcircuits of Layer 6 Interneurons in Rat Prefrontal Cortex

Layer-Specific Inhibitory Microcircuits of Layer 6 Interneurons in Rat Prefrontal Cortex

Filopodyan: An open-source pipeline for the analysis of filopodia

The tumor promoter‐activated protein kinase Cs are a system for regulating filopodia

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