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Sandhya P. Koushika - One of the best experts on this subject based on the ideXlab platform.

  • Overexpression of LRK-1 suppresses the SVP-related trafficking defects seen in unc-16.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) SNB-1::GFP is mis-localized into the dendritic compartment of the amphid sensory neurons in unc-16 (tb109) with the defect being suppressed in unc-16 (tb109); kmEx1180 (LRK-1::FLAG) animals. (b) Dual colour imaging and quantitation from Kymograph analysis shows that Golgi enzyme Man-II::mCherry is not co-transported in the same compartment along with synaptic vesicle marker GFP::RAB-3 into the PLM neuronal processes of unc-16; kmEx1180 animals. n ≥ 10 animals (c) Dual colour imaging and quantitation from Kymograph analysis shows that synaptic vesicle proteins mCherry::RAB-3 and SNB-1::GFP predominantly travel in the same compartment along the PLM neuronal process in unc-16 (tb109); tbIs259 (LRK-1::FLAG) animals. n ≥ 10 animals (d) and (e) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows reduced presence of long moving compartments in unc-16; kmEx1180 animals. n ≥ 10 animals, particles measured > 200 per genotype (f) LRK-1::VENUS localization is altered in neuronal cell bodies of unc-16 (tb109) mutants suggesting that UNC-16 regulates Golgi-localization of LRK-1. n ≥ 10 animals. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In image panel, scale bar represents 10μm.

  • lrk-1 mutants show a subset of mis-trafficking defects that is observed in unc-16 mutants.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) SNB-1::GFP is mis-localized into the dendritic compartment of the amphid sensory neuron in lrk-1(km17) and lrk-1(km17); unc-16 (tb109) animals, similar to unc-16 (tb109) animals. (b) Dual colour imaging and quantitation from Kymograph analysis shows that Golgi enzyme Man-II::mCherry is not co-transported in the same compartment along with synaptic vesicle marker GFP::RAB-3 in the PLM neuronal processes of multiple alleles of lrk-1 (km17 and km41), unlike in unc-16 (tb109) and lrk-1(km17); unc-16(tb109) animals. n ≥ 10 animals per genotype. (c) Dual colour imaging and quantitation from Kymograph analysis shows that synaptic vesicle proteins mCherry::RAB-3 and SNB-1::GFP mostly travel in different compartments along the PLM neuronal process in multiple alleles of lrk-1 (km17 and km41), unc-16 (tb109) and lrk-1(km17); unc-16(tb109) animals. n ≥ 10 animals per genotype (d) and (e) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows increased presence of long moving compartments (indicated by red arrowheads) in multiple alleles of lrk-1 (km17 and km41) and lrk-1(km17); unc-16(tb109) double mutants. n ≥ 10 animals, particles measured > 300 compartments per genotype. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In image panel, scale bar represents 10μm.

  • unc-101 and apb-3 acts downstream of unc-16 and lrk-1 to regulate size and composition of SVP transport carriers respectively.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) SNB-1::GFP is mis-localized into the dendritic compartment of the amphid sensory neurons in unc-16 (tb109) and unc-101 (m1); unc-16 (tb109) but not in wild type or unc-101 (m1) animals (b) Dual colour imaging and quantitation from Kymograph analysis shows Golgi enzyme Man-II::mCherry is co-transported with synaptic vesicle marker GFP::RAB-3 into the PLM neuronal process in unc-101; unc-16 but not in unc-101 (m1), unc-101 (m1), lrk-1 (km17) and apb-3 (ok429) animals. n ≥ 10 animals (c) Dual colour imaging and quantitation from Kymograph analysis shows co-transport of GFP::RAB-3 and SNB-1::GFP in the PLM neuronal process reduces in apb-3 (ok429), apb-3 (ok429), lrk-1 (km17), apb-3 (ok429); unc-16 (tb109) and in apb-3 (ok429); unc-16 (tb109); tbIs259 (LRK-1::FLAG) but not in unc-16 (tb109); tbIs259 (LRK-1::FLAG) or unc-101 (m1) mutants. n ≥ 10 (LRK-1::FLAG) animals (d) and (e) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows increased presence of long moving compartments (indicated by red arrowheads) in unc-101 (m1) and in unc-101 (m1); unc-16 (tb109); kmEX1180 (LRK-1::FLAG) animals but not in apb-3 (ok429) animals. n ≥ 10 animals, particles measured > 200 compartments per genotype. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In image panel, scale bar represents 10μm.

  • UNC-16 regulates the size of SVP transport carriers.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows increased presence of long moving compartments (indicated by red arrowheads) in unc-16 (tb109) and unc-16 (ju146) animals. n ≥ 10 animals, particles measured > 300 compartments per genotype (b) Electron micrographs of ventral cord section, representing non-synaptic regions, shows the presence of vesicular structures (indicated by arrowheads). A diagrammatic representation of the section is shown below the EM images to outline the large vesicular structures seen in unc-16 (tb109) mutants compared to wild type animals. n = 5 animals for wild type and 9 animals for tb109. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In electron micrograph scale bar represents 0.1μm.

  • UNC-16 is essential to exclude Golgi enzymes from SVP transport carriers and to include certain SVPs in the same transport carrier.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) UNC-16::GFP co-localizes with Golgi markers RUND-1::tagRFP and Mannosidase-II::mCherry and can be observed as 1–3 puncta in the cell bodies of PLM neurons. (b) and (d) Dual colour imaging and quantitation from Kymograph analysis shows Golgi enzyme Man-II::mCherry is co-transported in the same compartment along with synaptic vesicle marker GFP::RAB-3 into the PLM neuronal processes in multiple unc-16 alleles (tb109 and e109). This mis-localization can be rescued by transgenic expression of wild type UNC-16 [kmEx1000 (T7::UNC-16)]. Yellow arrowheads indicate co-localization of both GFP::RAB-3 and MAN-II::mCherry. (c) and (e) Dual colour imaging and quantitation from Kymograph analysis shows the decreased incidence of synaptic vesicle proteins mCherry::RAB-3 and SNB-1::GFP travelling in the same compartment along the PLM neuronal process in multiple unc-16 alleles (tb109 and e109). Yellow arrowheads indicate co-localization of both mCherry::RAB-3 and SNB-1::GFP while red and green arrowheads represent either marker respectively. n ≥ 10 animals for wild type, tb109 and tb109; kmEx1000 and n = 6 for e109 genotypes. Scale Bar: In Kymographs, horizontal scale bar represents 5μm and vertical scale bar represent 40sec. In image panel, scale bar represents 10μm.

Bikash Choudhary - One of the best experts on this subject based on the ideXlab platform.

  • Overexpression of LRK-1 suppresses the SVP-related trafficking defects seen in unc-16.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) SNB-1::GFP is mis-localized into the dendritic compartment of the amphid sensory neurons in unc-16 (tb109) with the defect being suppressed in unc-16 (tb109); kmEx1180 (LRK-1::FLAG) animals. (b) Dual colour imaging and quantitation from Kymograph analysis shows that Golgi enzyme Man-II::mCherry is not co-transported in the same compartment along with synaptic vesicle marker GFP::RAB-3 into the PLM neuronal processes of unc-16; kmEx1180 animals. n ≥ 10 animals (c) Dual colour imaging and quantitation from Kymograph analysis shows that synaptic vesicle proteins mCherry::RAB-3 and SNB-1::GFP predominantly travel in the same compartment along the PLM neuronal process in unc-16 (tb109); tbIs259 (LRK-1::FLAG) animals. n ≥ 10 animals (d) and (e) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows reduced presence of long moving compartments in unc-16; kmEx1180 animals. n ≥ 10 animals, particles measured > 200 per genotype (f) LRK-1::VENUS localization is altered in neuronal cell bodies of unc-16 (tb109) mutants suggesting that UNC-16 regulates Golgi-localization of LRK-1. n ≥ 10 animals. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In image panel, scale bar represents 10μm.

  • lrk-1 mutants show a subset of mis-trafficking defects that is observed in unc-16 mutants.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) SNB-1::GFP is mis-localized into the dendritic compartment of the amphid sensory neuron in lrk-1(km17) and lrk-1(km17); unc-16 (tb109) animals, similar to unc-16 (tb109) animals. (b) Dual colour imaging and quantitation from Kymograph analysis shows that Golgi enzyme Man-II::mCherry is not co-transported in the same compartment along with synaptic vesicle marker GFP::RAB-3 in the PLM neuronal processes of multiple alleles of lrk-1 (km17 and km41), unlike in unc-16 (tb109) and lrk-1(km17); unc-16(tb109) animals. n ≥ 10 animals per genotype. (c) Dual colour imaging and quantitation from Kymograph analysis shows that synaptic vesicle proteins mCherry::RAB-3 and SNB-1::GFP mostly travel in different compartments along the PLM neuronal process in multiple alleles of lrk-1 (km17 and km41), unc-16 (tb109) and lrk-1(km17); unc-16(tb109) animals. n ≥ 10 animals per genotype (d) and (e) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows increased presence of long moving compartments (indicated by red arrowheads) in multiple alleles of lrk-1 (km17 and km41) and lrk-1(km17); unc-16(tb109) double mutants. n ≥ 10 animals, particles measured > 300 compartments per genotype. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In image panel, scale bar represents 10μm.

  • unc-101 and apb-3 acts downstream of unc-16 and lrk-1 to regulate size and composition of SVP transport carriers respectively.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) SNB-1::GFP is mis-localized into the dendritic compartment of the amphid sensory neurons in unc-16 (tb109) and unc-101 (m1); unc-16 (tb109) but not in wild type or unc-101 (m1) animals (b) Dual colour imaging and quantitation from Kymograph analysis shows Golgi enzyme Man-II::mCherry is co-transported with synaptic vesicle marker GFP::RAB-3 into the PLM neuronal process in unc-101; unc-16 but not in unc-101 (m1), unc-101 (m1), lrk-1 (km17) and apb-3 (ok429) animals. n ≥ 10 animals (c) Dual colour imaging and quantitation from Kymograph analysis shows co-transport of GFP::RAB-3 and SNB-1::GFP in the PLM neuronal process reduces in apb-3 (ok429), apb-3 (ok429), lrk-1 (km17), apb-3 (ok429); unc-16 (tb109) and in apb-3 (ok429); unc-16 (tb109); tbIs259 (LRK-1::FLAG) but not in unc-16 (tb109); tbIs259 (LRK-1::FLAG) or unc-101 (m1) mutants. n ≥ 10 (LRK-1::FLAG) animals (d) and (e) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows increased presence of long moving compartments (indicated by red arrowheads) in unc-101 (m1) and in unc-101 (m1); unc-16 (tb109); kmEX1180 (LRK-1::FLAG) animals but not in apb-3 (ok429) animals. n ≥ 10 animals, particles measured > 200 compartments per genotype. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In image panel, scale bar represents 10μm.

  • UNC-16 regulates the size of SVP transport carriers.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows increased presence of long moving compartments (indicated by red arrowheads) in unc-16 (tb109) and unc-16 (ju146) animals. n ≥ 10 animals, particles measured > 300 compartments per genotype (b) Electron micrographs of ventral cord section, representing non-synaptic regions, shows the presence of vesicular structures (indicated by arrowheads). A diagrammatic representation of the section is shown below the EM images to outline the large vesicular structures seen in unc-16 (tb109) mutants compared to wild type animals. n = 5 animals for wild type and 9 animals for tb109. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In electron micrograph scale bar represents 0.1μm.

  • UNC-16 is essential to exclude Golgi enzymes from SVP transport carriers and to include certain SVPs in the same transport carrier.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) UNC-16::GFP co-localizes with Golgi markers RUND-1::tagRFP and Mannosidase-II::mCherry and can be observed as 1–3 puncta in the cell bodies of PLM neurons. (b) and (d) Dual colour imaging and quantitation from Kymograph analysis shows Golgi enzyme Man-II::mCherry is co-transported in the same compartment along with synaptic vesicle marker GFP::RAB-3 into the PLM neuronal processes in multiple unc-16 alleles (tb109 and e109). This mis-localization can be rescued by transgenic expression of wild type UNC-16 [kmEx1000 (T7::UNC-16)]. Yellow arrowheads indicate co-localization of both GFP::RAB-3 and MAN-II::mCherry. (c) and (e) Dual colour imaging and quantitation from Kymograph analysis shows the decreased incidence of synaptic vesicle proteins mCherry::RAB-3 and SNB-1::GFP travelling in the same compartment along the PLM neuronal process in multiple unc-16 alleles (tb109 and e109). Yellow arrowheads indicate co-localization of both mCherry::RAB-3 and SNB-1::GFP while red and green arrowheads represent either marker respectively. n ≥ 10 animals for wild type, tb109 and tb109; kmEx1000 and n = 6 for e109 genotypes. Scale Bar: In Kymographs, horizontal scale bar represents 5μm and vertical scale bar represent 40sec. In image panel, scale bar represents 10μm.

Oliver Welzel - One of the best experts on this subject based on the ideXlab platform.

  • A fast and robust method for automated analysis of axonal transport
    European Biophysics Journal, 2011
    Co-Authors: Oliver Welzel, Johannes Kornhuber, Jutta Knörr, Armin M. Stroebel, Teja W. Groemer
    Abstract:

    Cargo movement along axons and dendrites is indispensable for the survival and maintenance of neuronal networks. Key parameters of this transport such as particle velocities and pausing times are often studied using Kymograph construction, which converts the transport along a line of interest from a time-lapse movie into a position versus time image. Here we present a method for the automatic analysis of such Kymographs based on the Hough transform, which is a robust and fast technique to extract lines from images. The applicability of the method was tested on simulated Kymograph images and real data from axonal transport of synaptophysin and tetanus toxin as well as the velocity analysis of synaptic vesicle sharing between adjacent synapses in hippocampal neurons. Efficiency analysis revealed that the algorithm is able to detect a wide range of velocities and can be used at low signal-to-noise ratios. The present work enables the quantification of axonal transport parameters with high throughput with no a priori assumptions and minimal human intervention.

  • Determination of axonal transport velocities via image cross- and autocorrelation
    European Biophysics Journal, 2009
    Co-Authors: Oliver Welzel, Daniel Boening, Armin Stroebel, Udo Reulbach, Jurgen Klingauf, Johannes Kornhuber, Teja Wolfgang Groemer
    Abstract:

    On their way to the synapse and back, neuronal proteins are carried in cargo vesicles along axons and dendrites. Here, we demonstrate that the key parameters of axonal transport, i.e., particle velocities and pausing times can be read out from CCD-camera images automatically. In the present study, this is achieved via cross- and autocorrelation of Kymograph columns. The applicability of the method was measured on simulated Kymographs and data from axonal transport timeseries of mRFP-labeled synaptophysin. In comparing outcomes of velocity determinations via a performance parameter that is analogous to the signal-to-noise ratio (SNR) definition, we find that outcomes are dependent on sampling, particle numbers and signal to noise of the Kymograph. Autocorrelation of individual columns allows exact determination of pausing time populations. In contrast to manual tracking, correlation does not require experience, a priori assumptions or disentangling of individual particle trajectories and can operate at low SNR.

Ken Nguyen - One of the best experts on this subject based on the ideXlab platform.

  • Overexpression of LRK-1 suppresses the SVP-related trafficking defects seen in unc-16.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) SNB-1::GFP is mis-localized into the dendritic compartment of the amphid sensory neurons in unc-16 (tb109) with the defect being suppressed in unc-16 (tb109); kmEx1180 (LRK-1::FLAG) animals. (b) Dual colour imaging and quantitation from Kymograph analysis shows that Golgi enzyme Man-II::mCherry is not co-transported in the same compartment along with synaptic vesicle marker GFP::RAB-3 into the PLM neuronal processes of unc-16; kmEx1180 animals. n ≥ 10 animals (c) Dual colour imaging and quantitation from Kymograph analysis shows that synaptic vesicle proteins mCherry::RAB-3 and SNB-1::GFP predominantly travel in the same compartment along the PLM neuronal process in unc-16 (tb109); tbIs259 (LRK-1::FLAG) animals. n ≥ 10 animals (d) and (e) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows reduced presence of long moving compartments in unc-16; kmEx1180 animals. n ≥ 10 animals, particles measured > 200 per genotype (f) LRK-1::VENUS localization is altered in neuronal cell bodies of unc-16 (tb109) mutants suggesting that UNC-16 regulates Golgi-localization of LRK-1. n ≥ 10 animals. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In image panel, scale bar represents 10μm.

  • lrk-1 mutants show a subset of mis-trafficking defects that is observed in unc-16 mutants.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) SNB-1::GFP is mis-localized into the dendritic compartment of the amphid sensory neuron in lrk-1(km17) and lrk-1(km17); unc-16 (tb109) animals, similar to unc-16 (tb109) animals. (b) Dual colour imaging and quantitation from Kymograph analysis shows that Golgi enzyme Man-II::mCherry is not co-transported in the same compartment along with synaptic vesicle marker GFP::RAB-3 in the PLM neuronal processes of multiple alleles of lrk-1 (km17 and km41), unlike in unc-16 (tb109) and lrk-1(km17); unc-16(tb109) animals. n ≥ 10 animals per genotype. (c) Dual colour imaging and quantitation from Kymograph analysis shows that synaptic vesicle proteins mCherry::RAB-3 and SNB-1::GFP mostly travel in different compartments along the PLM neuronal process in multiple alleles of lrk-1 (km17 and km41), unc-16 (tb109) and lrk-1(km17); unc-16(tb109) animals. n ≥ 10 animals per genotype (d) and (e) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows increased presence of long moving compartments (indicated by red arrowheads) in multiple alleles of lrk-1 (km17 and km41) and lrk-1(km17); unc-16(tb109) double mutants. n ≥ 10 animals, particles measured > 300 compartments per genotype. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In image panel, scale bar represents 10μm.

  • unc-101 and apb-3 acts downstream of unc-16 and lrk-1 to regulate size and composition of SVP transport carriers respectively.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) SNB-1::GFP is mis-localized into the dendritic compartment of the amphid sensory neurons in unc-16 (tb109) and unc-101 (m1); unc-16 (tb109) but not in wild type or unc-101 (m1) animals (b) Dual colour imaging and quantitation from Kymograph analysis shows Golgi enzyme Man-II::mCherry is co-transported with synaptic vesicle marker GFP::RAB-3 into the PLM neuronal process in unc-101; unc-16 but not in unc-101 (m1), unc-101 (m1), lrk-1 (km17) and apb-3 (ok429) animals. n ≥ 10 animals (c) Dual colour imaging and quantitation from Kymograph analysis shows co-transport of GFP::RAB-3 and SNB-1::GFP in the PLM neuronal process reduces in apb-3 (ok429), apb-3 (ok429), lrk-1 (km17), apb-3 (ok429); unc-16 (tb109) and in apb-3 (ok429); unc-16 (tb109); tbIs259 (LRK-1::FLAG) but not in unc-16 (tb109); tbIs259 (LRK-1::FLAG) or unc-101 (m1) mutants. n ≥ 10 (LRK-1::FLAG) animals (d) and (e) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows increased presence of long moving compartments (indicated by red arrowheads) in unc-101 (m1) and in unc-101 (m1); unc-16 (tb109); kmEX1180 (LRK-1::FLAG) animals but not in apb-3 (ok429) animals. n ≥ 10 animals, particles measured > 200 compartments per genotype. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In image panel, scale bar represents 10μm.

  • UNC-16 regulates the size of SVP transport carriers.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows increased presence of long moving compartments (indicated by red arrowheads) in unc-16 (tb109) and unc-16 (ju146) animals. n ≥ 10 animals, particles measured > 300 compartments per genotype (b) Electron micrographs of ventral cord section, representing non-synaptic regions, shows the presence of vesicular structures (indicated by arrowheads). A diagrammatic representation of the section is shown below the EM images to outline the large vesicular structures seen in unc-16 (tb109) mutants compared to wild type animals. n = 5 animals for wild type and 9 animals for tb109. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In electron micrograph scale bar represents 0.1μm.

  • UNC-16 is essential to exclude Golgi enzymes from SVP transport carriers and to include certain SVPs in the same transport carrier.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) UNC-16::GFP co-localizes with Golgi markers RUND-1::tagRFP and Mannosidase-II::mCherry and can be observed as 1–3 puncta in the cell bodies of PLM neurons. (b) and (d) Dual colour imaging and quantitation from Kymograph analysis shows Golgi enzyme Man-II::mCherry is co-transported in the same compartment along with synaptic vesicle marker GFP::RAB-3 into the PLM neuronal processes in multiple unc-16 alleles (tb109 and e109). This mis-localization can be rescued by transgenic expression of wild type UNC-16 [kmEx1000 (T7::UNC-16)]. Yellow arrowheads indicate co-localization of both GFP::RAB-3 and MAN-II::mCherry. (c) and (e) Dual colour imaging and quantitation from Kymograph analysis shows the decreased incidence of synaptic vesicle proteins mCherry::RAB-3 and SNB-1::GFP travelling in the same compartment along the PLM neuronal process in multiple unc-16 alleles (tb109 and e109). Yellow arrowheads indicate co-localization of both mCherry::RAB-3 and SNB-1::GFP while red and green arrowheads represent either marker respectively. n ≥ 10 animals for wild type, tb109 and tb109; kmEx1000 and n = 6 for e109 genotypes. Scale Bar: In Kymographs, horizontal scale bar represents 5μm and vertical scale bar represent 40sec. In image panel, scale bar represents 10μm.

Madhushree Kamak - One of the best experts on this subject based on the ideXlab platform.

  • Overexpression of LRK-1 suppresses the SVP-related trafficking defects seen in unc-16.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) SNB-1::GFP is mis-localized into the dendritic compartment of the amphid sensory neurons in unc-16 (tb109) with the defect being suppressed in unc-16 (tb109); kmEx1180 (LRK-1::FLAG) animals. (b) Dual colour imaging and quantitation from Kymograph analysis shows that Golgi enzyme Man-II::mCherry is not co-transported in the same compartment along with synaptic vesicle marker GFP::RAB-3 into the PLM neuronal processes of unc-16; kmEx1180 animals. n ≥ 10 animals (c) Dual colour imaging and quantitation from Kymograph analysis shows that synaptic vesicle proteins mCherry::RAB-3 and SNB-1::GFP predominantly travel in the same compartment along the PLM neuronal process in unc-16 (tb109); tbIs259 (LRK-1::FLAG) animals. n ≥ 10 animals (d) and (e) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows reduced presence of long moving compartments in unc-16; kmEx1180 animals. n ≥ 10 animals, particles measured > 200 per genotype (f) LRK-1::VENUS localization is altered in neuronal cell bodies of unc-16 (tb109) mutants suggesting that UNC-16 regulates Golgi-localization of LRK-1. n ≥ 10 animals. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In image panel, scale bar represents 10μm.

  • lrk-1 mutants show a subset of mis-trafficking defects that is observed in unc-16 mutants.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) SNB-1::GFP is mis-localized into the dendritic compartment of the amphid sensory neuron in lrk-1(km17) and lrk-1(km17); unc-16 (tb109) animals, similar to unc-16 (tb109) animals. (b) Dual colour imaging and quantitation from Kymograph analysis shows that Golgi enzyme Man-II::mCherry is not co-transported in the same compartment along with synaptic vesicle marker GFP::RAB-3 in the PLM neuronal processes of multiple alleles of lrk-1 (km17 and km41), unlike in unc-16 (tb109) and lrk-1(km17); unc-16(tb109) animals. n ≥ 10 animals per genotype. (c) Dual colour imaging and quantitation from Kymograph analysis shows that synaptic vesicle proteins mCherry::RAB-3 and SNB-1::GFP mostly travel in different compartments along the PLM neuronal process in multiple alleles of lrk-1 (km17 and km41), unc-16 (tb109) and lrk-1(km17); unc-16(tb109) animals. n ≥ 10 animals per genotype (d) and (e) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows increased presence of long moving compartments (indicated by red arrowheads) in multiple alleles of lrk-1 (km17 and km41) and lrk-1(km17); unc-16(tb109) double mutants. n ≥ 10 animals, particles measured > 300 compartments per genotype. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In image panel, scale bar represents 10μm.

  • unc-101 and apb-3 acts downstream of unc-16 and lrk-1 to regulate size and composition of SVP transport carriers respectively.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) SNB-1::GFP is mis-localized into the dendritic compartment of the amphid sensory neurons in unc-16 (tb109) and unc-101 (m1); unc-16 (tb109) but not in wild type or unc-101 (m1) animals (b) Dual colour imaging and quantitation from Kymograph analysis shows Golgi enzyme Man-II::mCherry is co-transported with synaptic vesicle marker GFP::RAB-3 into the PLM neuronal process in unc-101; unc-16 but not in unc-101 (m1), unc-101 (m1), lrk-1 (km17) and apb-3 (ok429) animals. n ≥ 10 animals (c) Dual colour imaging and quantitation from Kymograph analysis shows co-transport of GFP::RAB-3 and SNB-1::GFP in the PLM neuronal process reduces in apb-3 (ok429), apb-3 (ok429), lrk-1 (km17), apb-3 (ok429); unc-16 (tb109) and in apb-3 (ok429); unc-16 (tb109); tbIs259 (LRK-1::FLAG) but not in unc-16 (tb109); tbIs259 (LRK-1::FLAG) or unc-101 (m1) mutants. n ≥ 10 (LRK-1::FLAG) animals (d) and (e) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows increased presence of long moving compartments (indicated by red arrowheads) in unc-101 (m1) and in unc-101 (m1); unc-16 (tb109); kmEX1180 (LRK-1::FLAG) animals but not in apb-3 (ok429) animals. n ≥ 10 animals, particles measured > 200 compartments per genotype. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In image panel, scale bar represents 10μm.

  • UNC-16 regulates the size of SVP transport carriers.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows increased presence of long moving compartments (indicated by red arrowheads) in unc-16 (tb109) and unc-16 (ju146) animals. n ≥ 10 animals, particles measured > 300 compartments per genotype (b) Electron micrographs of ventral cord section, representing non-synaptic regions, shows the presence of vesicular structures (indicated by arrowheads). A diagrammatic representation of the section is shown below the EM images to outline the large vesicular structures seen in unc-16 (tb109) mutants compared to wild type animals. n = 5 animals for wild type and 9 animals for tb109. Scale Bar: In Kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In electron micrograph scale bar represents 0.1μm.

  • UNC-16 is essential to exclude Golgi enzymes from SVP transport carriers and to include certain SVPs in the same transport carrier.
    2017
    Co-Authors: Bikash Choudhary, Madhushree Kamak, Neena Ratnakaran, Jitendra Kumar, Anjali Awasthi, Ken Nguyen, Kunihiro Matsumoto, Naoki Hisamoto, Sandhya P. Koushika
    Abstract:

    (a) UNC-16::GFP co-localizes with Golgi markers RUND-1::tagRFP and Mannosidase-II::mCherry and can be observed as 1–3 puncta in the cell bodies of PLM neurons. (b) and (d) Dual colour imaging and quantitation from Kymograph analysis shows Golgi enzyme Man-II::mCherry is co-transported in the same compartment along with synaptic vesicle marker GFP::RAB-3 into the PLM neuronal processes in multiple unc-16 alleles (tb109 and e109). This mis-localization can be rescued by transgenic expression of wild type UNC-16 [kmEx1000 (T7::UNC-16)]. Yellow arrowheads indicate co-localization of both GFP::RAB-3 and MAN-II::mCherry. (c) and (e) Dual colour imaging and quantitation from Kymograph analysis shows the decreased incidence of synaptic vesicle proteins mCherry::RAB-3 and SNB-1::GFP travelling in the same compartment along the PLM neuronal process in multiple unc-16 alleles (tb109 and e109). Yellow arrowheads indicate co-localization of both mCherry::RAB-3 and SNB-1::GFP while red and green arrowheads represent either marker respectively. n ≥ 10 animals for wild type, tb109 and tb109; kmEx1000 and n = 6 for e109 genotypes. Scale Bar: In Kymographs, horizontal scale bar represents 5μm and vertical scale bar represent 40sec. In image panel, scale bar represents 10μm.

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