188 lines
34 KiB
XML
188 lines
34 KiB
XML
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<mods:title id="BC2C9B857D57BF26C1AA6F7B5462CEE4">Immunocytochemical Analysis of α-Tubulin Distribution Before and After Rapid Axopodial Contraction in the Centrohelid Raphidocystis contractilis</mods:title>
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<mods:namePart id="469BA09C1C22681EA3E912E267510907">Ikeda, Risa</mods:namePart>
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<mods:namePart id="25E57D967F14D121E350505F92F30EF1">Kurokawa, Miki</mods:namePart>
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<mods:namePart id="EE8FF4B0A1F0966D8E8FA5E8FF4A6D16">Murai, Momoka</mods:namePart>
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<mods:namePart id="C575CB22C80121966C60104AE43F1AB4">Ando, Motonori</mods:namePart>
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<treatment id="03A98783675CFFF8140D8AC9FD5AFBA7" LSID="urn:lsid:plazi:treatment:03A98783675CFFF8140D8AC9FD5AFBA7" httpUri="http://treatment.plazi.org/id/03A98783675CFFF8140D8AC9FD5AFBA7" lastPageId="7" lastPageNumber="5" pageId="2" pageNumber="3">
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<subSubSection id="C31A651E675CFFFD140D8AC9FACCFC88" box="[816,1399,760,787]" pageId="2" pageNumber="3" type="nomenclature">
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<paragraph id="8BBF3695675CFFFD140D8AC9FACCFC88" blockId="2.[816,1399,760,787]" box="[816,1399,760,787]" pageId="2" pageNumber="3">
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<heading id="D0F781F9675CFFFD140D8AC9FACCFC88" box="[816,1399,760,787]" fontSize="11" level="2" pageId="2" pageNumber="3" reason="5">
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<emphasis id="B974EA87675CFFFD140D8AC9FACCFC88" bold="true" box="[816,1399,760,787]" pageId="2" pageNumber="3">
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Morphological characteristics of
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<taxonomicName id="4C004D16675CFFFD13F08AC8FACCFC88" authority="Kinoshita, Suzaki, Shigenaka & Sugiyama, 1995" authorityName="Kinoshita, Suzaki, Shigenaka & Sugiyama" authorityYear="1995" box="[1229,1399,760,787]" family="Raphidiophryidae" genus="Raphidiophrys" kingdom="Chromista" order="Centrohelida" pageId="2" pageNumber="3" phylum="Heliozoa" rank="species" species="contractilis">
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<emphasis id="B974EA87675CFFFD13F08AC8FACCFC88" bold="true" box="[1229,1399,760,787]" italics="true" pageId="2" pageNumber="3">R. contractilis</emphasis>
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<paragraph id="8BBF3695675CFFFD146B8B1BFB1EF9D9" blockId="2.[816,1463,810,1602]" pageId="2" pageNumber="3">
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Rapid axopodial contraction in
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<taxonomicName id="4C004D16675CFFFD13E88B1AFAC4FCDF" box="[1237,1407,810,836]" family="Raphidiophryidae" genus="Raphidiophrys" kingdom="Chromista" order="Centrohelida" pageId="2" pageNumber="3" phylum="Heliozoa" rank="species" species="contractilis">
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<emphasis id="B974EA87675CFFFD13E88B1AFAC4FCDF" box="[1237,1407,810,836]" italics="true" pageId="2" pageNumber="3">R. contractilis</emphasis>
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</taxonomicName>
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was examined using video microscopy (
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<figureCitation id="133B2A10675CFFFD13E98B7CFAA2FCFC" box="[1236,1305,845,871]" captionStart="Fig" captionStartId="3.[125,160,917,939]" captionTargetBox="[125,1456,225,887]" captionTargetId="figure-7@3.[125,1456,225,887]" captionTargetPageId="3" captionText="Fig. 1. (A, B) Rapid axopodial contraction induced by mechanical stimulation in R. contractilis. Images (A) before and (B) after rapid axopodial contraction. Arrowheads indicate kinetocysts. Note the synchronized retraction of all axopodia and the apparent increases in the widths of contracted axopodia relative to the features observed before the onset of axopodial contraction (an arrow). Scale bar: 20 µm (A, B)." figureDoi="http://doi.org/10.5281/zenodo.10994457" httpUri="https://zenodo.org/record/10994457/files/figure.png" pageId="2" pageNumber="3">Fig. 1</figureCitation>
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).
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<taxonomicName id="4C004D16675CFFFD120F8B7FFCE7FC11" family="Raphidiophryidae" genus="Raphidiophrys" kingdom="Chromista" order="Centrohelida" pageId="2" pageNumber="3" phylum="Heliozoa" rank="species" species="contractilis">
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<emphasis id="B974EA87675CFFFD120F8B7FFCE7FC11" italics="true" pageId="2" pageNumber="3">R. contractilis</emphasis>
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</taxonomicName>
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has a spherical cell body surrounded by several radiating axopodia (
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<figureCitation id="133B2A10675CFFFD131D8BA2FBC4FC36" box="[1056,1151,915,941]" captionStart="Fig" captionStartId="3.[125,160,917,939]" captionTargetBox="[125,1456,225,887]" captionTargetId="figure-7@3.[125,1456,225,887]" captionTargetPageId="3" captionText="Fig. 1. (A, B) Rapid axopodial contraction induced by mechanical stimulation in R. contractilis. Images (A) before and (B) after rapid axopodial contraction. Arrowheads indicate kinetocysts. Note the synchronized retraction of all axopodia and the apparent increases in the widths of contracted axopodia relative to the features observed before the onset of axopodial contraction (an arrow). Scale bar: 20 µm (A, B)." figureDoi="http://doi.org/10.5281/zenodo.10994457" httpUri="https://zenodo.org/record/10994457/files/figure.png" pageId="2" pageNumber="3">Fig. 1A</figureCitation>
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). These axopodia have an average length of 60 µm and a maximum length exceeding 100 µm. Each axopodium contains granular kinetocysts that participate in food capture (
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<figureCitation id="133B2A10675CFFFD121A8BCAFA3EFB8E" box="[1319,1413,1019,1045]" captionStart="Fig" captionStartId="3.[125,160,917,939]" captionTargetBox="[125,1456,225,887]" captionTargetId="figure-7@3.[125,1456,225,887]" captionTargetPageId="3" captionText="Fig. 1. (A, B) Rapid axopodial contraction induced by mechanical stimulation in R. contractilis. Images (A) before and (B) after rapid axopodial contraction. Arrowheads indicate kinetocysts. Note the synchronized retraction of all axopodia and the apparent increases in the widths of contracted axopodia relative to the features observed before the onset of axopodial contraction (an arrow). Scale bar: 20 µm (A, B)." figureDoi="http://doi.org/10.5281/zenodo.10994457" httpUri="https://zenodo.org/record/10994457/files/figure.png" pageId="2" pageNumber="3">Fig. 1A</figureCitation>
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,
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<emphasis id="B974EA87675CFFFD12AE8BCDFC1AFBA3" italics="true" pageId="2" pageNumber="3">arrowheads</emphasis>
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). Immediately after mechanical stimulation (see Methods), all axopodia retracted into the cell body at a less-than-video rate. The axopodial length was reduced to less than 10% of the initial length immediately after axopodial contraction (
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<figureCitation id="133B2A10675CFFFD13B98C98FB5AFB58" box="[1156,1249,1193,1219]" captionStart="Fig" captionStartId="3.[125,160,917,939]" captionTargetBox="[125,1456,225,887]" captionTargetId="figure-7@3.[125,1456,225,887]" captionTargetPageId="3" captionText="Fig. 1. (A, B) Rapid axopodial contraction induced by mechanical stimulation in R. contractilis. Images (A) before and (B) after rapid axopodial contraction. Arrowheads indicate kinetocysts. Note the synchronized retraction of all axopodia and the apparent increases in the widths of contracted axopodia relative to the features observed before the onset of axopodial contraction (an arrow). Scale bar: 20 µm (A, B)." figureDoi="http://doi.org/10.5281/zenodo.10994457" httpUri="https://zenodo.org/record/10994457/files/figure.png" pageId="2" pageNumber="3">Fig. 1B</figureCitation>
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). Simultaneously, the widths of the contracted axopodia appeared to increase compared with the widths before the onset of contraction (
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<figureCitation id="133B2A10675CFFFD14FB8D20FB9AFAB7" box="[966,1057,1297,1324]" captionStart="Fig" captionStartId="3.[125,160,917,939]" captionTargetBox="[125,1456,225,887]" captionTargetId="figure-7@3.[125,1456,225,887]" captionTargetPageId="3" captionText="Fig. 1. (A, B) Rapid axopodial contraction induced by mechanical stimulation in R. contractilis. Images (A) before and (B) after rapid axopodial contraction. Arrowheads indicate kinetocysts. Note the synchronized retraction of all axopodia and the apparent increases in the widths of contracted axopodia relative to the features observed before the onset of axopodial contraction (an arrow). Scale bar: 20 µm (A, B)." figureDoi="http://doi.org/10.5281/zenodo.10994457" httpUri="https://zenodo.org/record/10994457/files/figure.png" pageId="2" pageNumber="3">Fig. 1B</figureCitation>
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,
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<emphasis id="B974EA87675CFFFD130D8D23FBCCFAB0" box="[1072,1143,1298,1323]" italics="true" pageId="2" pageNumber="3">arrow</emphasis>
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). The microtubule orientation in the central and peripheral regions of the cells after rapid axopodial contraction was examined using conventional electron microscopy (
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<figureCitation id="133B2A10675CFFFD13ED8D4BFAA3FA0F" box="[1232,1304,1402,1428]" captionStart="Fig" captionStartId="3.[125,160,1860,1882]" captionTargetBox="[125,1456,1168,1830]" captionTargetId="figure-9@3.[125,1456,1168,1830]" captionTargetPageId="3" captionText="Fig. 2. (A, B) Fine structures associated with axopodial microtubules. (A) Centroplast in the center of the cell. (B) Cross-section of an axopo- dium in the peripheral region of the cell. Note that bundles of microtubules radiate from the centroplast (arrowheads) and that the axopodium comprises six microtubules. Scale bars: 500 nm (A), 100 nm (B)." figureDoi="http://doi.org/10.5281/zenodo.10994459" httpUri="https://zenodo.org/record/10994459/files/figure.png" pageId="2" pageNumber="3">Fig. 2</figureCitation>
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). The centroplast, a microtubule-organizing center presenting in the centrohelid heliozoa located at the center of each cell (
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<figureCitation id="133B2A10675CFFFD14048DD3FC23FA67" box="[825,920,1506,1532]" captionStart="Fig" captionStartId="3.[125,160,1860,1882]" captionTargetBox="[125,1456,1168,1830]" captionTargetId="figure-9@3.[125,1456,1168,1830]" captionTargetPageId="3" captionText="Fig. 2. (A, B) Fine structures associated with axopodial microtubules. (A) Centroplast in the center of the cell. (B) Cross-section of an axopo- dium in the peripheral region of the cell. Note that bundles of microtubules radiate from the centroplast (arrowheads) and that the axopodium comprises six microtubules. Scale bars: 500 nm (A), 100 nm (B)." figureDoi="http://doi.org/10.5281/zenodo.10994459" httpUri="https://zenodo.org/record/10994459/files/figure.png" pageId="2" pageNumber="3">Fig. 2A</figureCitation>
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). A cross-sectional analysis revealed that each axopodium comprised six microtubules in the peripheral region of the cell (
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<figureCitation id="133B2A10675CFFFD13068E19FB23F9D9" box="[1083,1176,1576,1602]" captionStart="Fig" captionStartId="3.[125,160,1860,1882]" captionTargetBox="[125,1456,1168,1830]" captionTargetId="figure-9@3.[125,1456,1168,1830]" captionTargetPageId="3" captionText="Fig. 2. (A, B) Fine structures associated with axopodial microtubules. (A) Centroplast in the center of the cell. (B) Cross-section of an axopo- dium in the peripheral region of the cell. Note that bundles of microtubules radiate from the centroplast (arrowheads) and that the axopodium comprises six microtubules. Scale bars: 500 nm (A), 100 nm (B)." figureDoi="http://doi.org/10.5281/zenodo.10994459" httpUri="https://zenodo.org/record/10994459/files/figure.png" pageId="2" pageNumber="3">Fig. 2B</figureCitation>
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).
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</paragraph>
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<paragraph id="8BBF3695675CFFFD140D8E68FB6AF9EF" blockId="2.[816,1233,1625,1652]" box="[816,1233,1625,1652]" pageId="2" pageNumber="3">
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<heading id="D0F781F9675CFFFD140D8E68FB6AF9EF" box="[816,1233,1625,1652]" fontSize="11" level="2" pageId="2" pageNumber="3" reason="5">
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<emphasis id="B974EA87675CFFFD140D8E68FB6AF9EF" bold="true" box="[816,1233,1625,1652]" pageId="2" pageNumber="3">Evaluation of fixation procedures</emphasis>
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</heading>
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</paragraph>
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<paragraph id="8BBF3695675CFFFB146B8EBDFE0CFA09" blockId="2.[816,1463,1676,1946]" lastBlockId="4.[132,779,1261,1946]" lastPageId="4" lastPageNumber="5" pageId="2" pageNumber="3">
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Next, we investigated the dependence of flow rate on the rapid axopodial contraction in
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<taxonomicName id="4C004D16675CFFFD12368E9EFA0CF953" box="[1291,1463,1710,1736]" family="Raphidiophryidae" genus="Raphidiophrys" kingdom="Chromista" order="Centrohelida" pageId="2" pageNumber="3" phylum="Heliozoa" rank="species" species="contractilis">
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<emphasis id="B974EA87675CFFFD12368E9EFA0CF953" box="[1291,1463,1710,1736]" italics="true" pageId="2" pageNumber="3">R. contractilis</emphasis>
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</taxonomicName>
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using a micro flow-through chamber. This chamber, equipped with a micro-syringe pump, was expected to mitigate the effect of shear stress against adherent cells during the injection of test solutions (
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<figureCitation id="133B2A10675CFFFD13D98F0BFA91F8CF" box="[1252,1322,1850,1876]" captionStart="Fig" captionStartId="4.[132,167,1062,1084]" captionTargetBox="[142,1463,225,1026]" captionTargetId="figure-374@4.[132,1463,225,1030]" captionTargetPageId="4" captionText="Fig. 3. Schematic illustration of the experimental setup. The fixative is injected into the cell suspension via a micro flow-through chamber with a syringe pump. Next, the cells are fixed by injecting the fixative at a rate below the threshold required for inducing rapid contraction. The cells are then observed under a microscope." figureDoi="http://doi.org/10.5281/zenodo.10994461" httpUri="https://zenodo.org/record/10994461/files/figure.png" pageId="2" pageNumber="3">Fig. 3</figureCitation>
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). We examined the effect of the flow rate on the rapid axopodial contraction by changing the flow rate from 0.5 to 500 μl/min. Notably, gentle perfusion with culture medium at a flow rate of <50 μl/min did not evoke rapid axopodial contraction. Therefore, in subsequent experiments, the test solutions were injected into the cell chamber at a flow rate of 12.5 μl/min.
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</paragraph>
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<caption id="DF7F661D675DFFFC17408BA4FAC3FC79" ID-DOI="http://doi.org/10.5281/zenodo.10994457" ID-Zenodo-Dep="10994457" httpUri="https://zenodo.org/record/10994457/files/figure.png" pageId="3" pageNumber="4" startId="3.[125,160,917,939]" targetBox="[125,1456,225,887]" targetPageId="3" targetType="figure">
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<paragraph id="8BBF3695675DFFFC17408BA4FAC3FC79" blockId="3.[125,1456,917,995]" pageId="3" pageNumber="4">
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<emphasis id="B974EA87675DFFFC17408BA4FF07FC31" bold="true" box="[125,188,917,939]" pageId="3" pageNumber="4">Fig. 1.</emphasis>
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(A, B) Rapid axopodial contraction induced by mechanical stimulation in
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<emphasis id="B974EA87675DFFFC14458BA7FBBEFC31" box="[888,1029,917,939]" italics="true" pageId="3" pageNumber="4">
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<taxonomicName id="4C004D16675DFFFC14458BA7FBBAFC31" box="[888,1025,917,939]" family="Raphidiophryidae" genus="Raphidiophrys" kingdom="Chromista" order="Centrohelida" pageId="3" pageNumber="4" phylum="Heliozoa" rank="species" species="contractilis">R. contractilis</taxonomicName>
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.
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</emphasis>
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Images (A) before and (B) after rapid axopodial contraction.
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<emphasis id="B974EA87675DFFFC16238B80FE2AFC5D" box="[286,401,945,966]" italics="true" pageId="3" pageNumber="4">Arrowheads</emphasis>
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indicate kinetocysts. Note the synchronized retraction of all axopodia and the apparent increases in the widths of contracted axopodia relative to the features observed before the onset of axopodial contraction (
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<emphasis id="B974EA87675DFFFC131E8BFFFBC1FC78" box="[1059,1146,974,995]" italics="true" pageId="3" pageNumber="4">an arrow</emphasis>
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). Scale bar: 20 µm (A, B).
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</paragraph>
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</caption>
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<caption id="DF7F661D675DFFFC17408F75FD5BF809" ID-DOI="http://doi.org/10.5281/zenodo.10994459" ID-Zenodo-Dep="10994459" httpUri="https://zenodo.org/record/10994459/files/figure.png" pageId="3" pageNumber="4" startId="3.[125,160,1860,1882]" targetBox="[125,1456,1168,1830]" targetPageId="3" targetType="figure">
|
||
<paragraph id="8BBF3695675DFFFC17408F75FD5BF809" blockId="3.[125,1455,1860,1938]" pageId="3" pageNumber="4">
|
||
<emphasis id="B974EA87675DFFFC17408F75FF07F8C2" bold="true" box="[125,188,1860,1882]" pageId="3" pageNumber="4">Fig. 2.</emphasis>
|
||
(A, B) Fine structures associated with axopodial microtubules. (A) Centroplast in the center of the cell. (B) Cross-section of an axopodium in the peripheral region of the cell. Note that bundles of microtubules radiate from the centroplast (
|
||
<emphasis id="B974EA87675DFFFC13688F51FB7FF8EE" box="[1109,1220,1888,1909]" italics="true" pageId="3" pageNumber="4">arrowheads</emphasis>
|
||
) and that the axopodium comprises six microtubules. Scale bars: 500 nm (A), 100 nm (B).
|
||
</paragraph>
|
||
</caption>
|
||
<caption id="DF7F661D675AFFFB17B98C17FDEFFBEF" ID-DOI="http://doi.org/10.5281/zenodo.10994461" ID-Zenodo-Dep="10994461" httpUri="https://zenodo.org/record/10994461/files/figure.png" pageId="4" pageNumber="5" startId="4.[132,167,1062,1084]" targetBox="[142,1463,225,1026]" targetPageId="4" targetType="figure">
|
||
<paragraph id="8BBF3695675AFFFB17B98C17FDEFFBEF" blockId="4.[132,1466,1062,1140]" pageId="4" pageNumber="5">
|
||
<emphasis id="B974EA87675AFFFB17B98C17FF7FFBA0" bold="true" box="[132,196,1062,1084]" pageId="4" pageNumber="5">Fig. 3.</emphasis>
|
||
Schematic illustration of the experimental setup. The fixative is injected into the cell suspension via a micro flow-through chamber with a syringe pump. Next, the cells are fixed by injecting the fixative at a rate below the threshold required for inducing rapid contraction. The cells are then observed under a microscope.
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="8BBF3695675AFFFB17978DAAFAADFA2E" blockId="4.[132,779,1261,1946]" lastBlockId="4.[816,1463,1261,1462]" pageId="4" pageNumber="5">
|
||
Next, the effects of fixatives on the morphological appearances of the axopodia were examined using light microscopy (
|
||
<figureCitation id="133B2A10675AFFFB161D8DEEFED0FA62" box="[288,363,1503,1529]" captionStart="Fig" captionStartId="5.[125,160,1604,1626]" captionTargetBox="[125,1456,225,1563]" captionTargetId="figure-7@5.[125,1456,225,1563]" captionTargetPageId="5" captionText="Fig. 4. (A–D) Evaluation of the maintenance of the original lengths of axopodia in R. contractilis using different combinations of fixative and buffer. Cells were fixed with (A) 4% paraformaldehyde in phosphate buffer, (B) 4% paraformaldehyde in PHEM, (C) 0.2% glutaralde- hyde in phosphate buffer, or (D) 0.2% glutaraldehyde in PHEM. Note that only fixation with 0.2% glutaraldehyde in PHEM maintained the axopodial length. Scale bar: 20 µm." figureDoi="http://doi.org/10.5281/zenodo.10994463" httpUri="https://zenodo.org/record/10994463/files/figure.png" pageId="4" pageNumber="5">Fig. 4</figureCitation>
|
||
). The cells were fixed in solutions containing 4% paraformaldehyde or 0.2% glutaraldehyde in phosphate buffer or PHEM. Initially, the cells were fixed with 4% paraformaldehyde in phosphate buffer. Despite the status of this fixative as the most widely used in immunohistochemical applications, we found that 4% paraformaldehyde caused a reduction in the lengths of axopodia compared with the original lengths before fixation (
|
||
<figureCitation id="133B2A10675AFFFB169E8EC5FDBEF894" box="[419,517,1780,1807]" captionStart="Fig" captionStartId="5.[125,160,1604,1626]" captionTargetBox="[125,1456,225,1563]" captionTargetId="figure-7@5.[125,1456,225,1563]" captionTargetPageId="5" captionText="Fig. 4. (A–D) Evaluation of the maintenance of the original lengths of axopodia in R. contractilis using different combinations of fixative and buffer. Cells were fixed with (A) 4% paraformaldehyde in phosphate buffer, (B) 4% paraformaldehyde in PHEM, (C) 0.2% glutaralde- hyde in phosphate buffer, or (D) 0.2% glutaraldehyde in PHEM. Note that only fixation with 0.2% glutaraldehyde in PHEM maintained the axopodial length. Scale bar: 20 µm." figureDoi="http://doi.org/10.5281/zenodo.10994463" httpUri="https://zenodo.org/record/10994463/files/figure.png" pageId="4" pageNumber="5">Fig. 4A</figureCitation>
|
||
). A similar result was obtained when the cells were fixed with 4% paraformaldehyde in PHEM (
|
||
<figureCitation id="133B2A10675AFFFB16638F0BFE01F8CF" box="[350,442,1850,1876]" captionStart="Fig" captionStartId="5.[125,160,1604,1626]" captionTargetBox="[125,1456,225,1563]" captionTargetId="figure-7@5.[125,1456,225,1563]" captionTargetPageId="5" captionText="Fig. 4. (A–D) Evaluation of the maintenance of the original lengths of axopodia in R. contractilis using different combinations of fixative and buffer. Cells were fixed with (A) 4% paraformaldehyde in phosphate buffer, (B) 4% paraformaldehyde in PHEM, (C) 0.2% glutaralde- hyde in phosphate buffer, or (D) 0.2% glutaraldehyde in PHEM. Note that only fixation with 0.2% glutaraldehyde in PHEM maintained the axopodial length. Scale bar: 20 µm." figureDoi="http://doi.org/10.5281/zenodo.10994463" httpUri="https://zenodo.org/record/10994463/files/figure.png" pageId="4" pageNumber="5">Fig. 4B</figureCitation>
|
||
), and fluorescence images of α-tubulin labeling revealed the breakdown of axopodial microtubules within the contracted axopodia (data not shown). Second, the cells were fixed using 0.2% glutaraldehyde. The axopodial lengths were not maintained when the cells were fixed with 0.2% glutaraldehyde in phosphate buffer (
|
||
<figureCitation id="133B2A10675AFFFB13378D67FBD1FAEB" box="[1034,1130,1366,1392]" captionStart="Fig" captionStartId="5.[125,160,1604,1626]" captionTargetBox="[125,1456,225,1563]" captionTargetId="figure-7@5.[125,1456,225,1563]" captionTargetPageId="5" captionText="Fig. 4. (A–D) Evaluation of the maintenance of the original lengths of axopodia in R. contractilis using different combinations of fixative and buffer. Cells were fixed with (A) 4% paraformaldehyde in phosphate buffer, (B) 4% paraformaldehyde in PHEM, (C) 0.2% glutaralde- hyde in phosphate buffer, or (D) 0.2% glutaraldehyde in PHEM. Note that only fixation with 0.2% glutaraldehyde in PHEM maintained the axopodial length. Scale bar: 20 µm." figureDoi="http://doi.org/10.5281/zenodo.10994463" httpUri="https://zenodo.org/record/10994463/files/figure.png" pageId="4" pageNumber="5">Fig. 4C</figureCitation>
|
||
). Conversely, the axopodial lengths were maintained when the cells were fixed with 0.2% glutaraldehyde in PHEM (
|
||
<figureCitation id="133B2A10675AFFFB13978DAAFAB2FA2E" box="[1194,1289,1435,1461]" captionStart="Fig" captionStartId="5.[125,160,1604,1626]" captionTargetBox="[125,1456,225,1563]" captionTargetId="figure-7@5.[125,1456,225,1563]" captionTargetPageId="5" captionText="Fig. 4. (A–D) Evaluation of the maintenance of the original lengths of axopodia in R. contractilis using different combinations of fixative and buffer. Cells were fixed with (A) 4% paraformaldehyde in phosphate buffer, (B) 4% paraformaldehyde in PHEM, (C) 0.2% glutaralde- hyde in phosphate buffer, or (D) 0.2% glutaraldehyde in PHEM. Note that only fixation with 0.2% glutaraldehyde in PHEM maintained the axopodial length. Scale bar: 20 µm." figureDoi="http://doi.org/10.5281/zenodo.10994463" httpUri="https://zenodo.org/record/10994463/files/figure.png" pageId="4" pageNumber="5">Fig. 4D</figureCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BBF3695675AFFFB140D8DFCFBAEF991" blockId="4.[816,1463,1485,1546]" pageId="4" pageNumber="5">
|
||
<heading id="D0F781F9675AFFFB140D8DFCFBAEF991" centered="true" fontSize="11" level="2" pageId="4" pageNumber="5" reason="5">
|
||
<emphasis id="B974EA87675AFFFB140D8DFCFBAEF991" bold="true" pageId="4" pageNumber="5">Distribution of α-tubulin before and after rapid axopodial contraction</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BBF3695675AFFF8146B8E13FD5AFBA7" blockId="4.[816,1463,1570,1946]" lastBlockId="7.[125,771,851,1084]" lastPageId="7" lastPageNumber="8" pageId="4" pageNumber="5">
|
||
The cellular distribution of α-tubulin before and after rapid axopodial contraction was examined using confocal microscopy. The cells were fixed using 0.2% glutaraldehyde in PHEM. Positive signals corresponding to α-tubulin were detected along the fully extended axopodia in the absence of induced axopodial contraction (
|
||
<figureCitation id="133B2A10675AFFFB144E8EC5FC6CF894" box="[883,983,1780,1807]" captionStart="Fig" captionStartId="6.[132,167,1608,1630]" captionTargetBox="[136,1466,225,1562]" captionTargetId="figure-7@6.[136,1467,225,1563]" captionTargetPageId="6" captionText="Fig. 5. (A, B) The distribution of α-tubulin in the cell before rapid axopodial contraction. (A) Low-magnification images of a cell without the induction of rapid axopodial contraction.A projection image of the whole cell was constructed from 192 optical sections obtained at 0.5 µm intervals (left); the corresponding light micrograph is also shown (right). (B) High-magnification images of the same cell shown in (A). A projection image of the equatorial plane of the cell body constructed from 5 optical sections taken at 0.5 µm intervals, and the correspond- ing light micrograph. The asterisk indicates a centroplast. Note that positive signals were detected along the fully extended axopodia. Scale bars: 20 μm (A), 10 μm (B)." figureDoi="http://doi.org/10.5281/zenodo.10994465" httpUri="https://zenodo.org/record/10994465/files/figure.png" pageId="4" pageNumber="5">Fig. 5A</figureCitation>
|
||
). Notably, the positive signals radiated from the centroplast (
|
||
<figureCitation id="133B2A10675AFFFB13158F26FB39F8AA" box="[1064,1154,1814,1841]" captionStart="Fig" captionStartId="6.[132,167,1608,1630]" captionTargetBox="[136,1466,225,1562]" captionTargetId="figure-7@6.[136,1467,225,1563]" captionTargetPageId="6" captionText="Fig. 5. (A, B) The distribution of α-tubulin in the cell before rapid axopodial contraction. (A) Low-magnification images of a cell without the induction of rapid axopodial contraction.A projection image of the whole cell was constructed from 192 optical sections obtained at 0.5 µm intervals (left); the corresponding light micrograph is also shown (right). (B) High-magnification images of the same cell shown in (A). A projection image of the equatorial plane of the cell body constructed from 5 optical sections taken at 0.5 µm intervals, and the correspond- ing light micrograph. The asterisk indicates a centroplast. Note that positive signals were detected along the fully extended axopodia. Scale bars: 20 μm (A), 10 μm (B)." figureDoi="http://doi.org/10.5281/zenodo.10994465" httpUri="https://zenodo.org/record/10994465/files/figure.png" pageId="4" pageNumber="5">Fig. 5B</figureCitation>
|
||
). A detailed observation of the extended axopodia in the equatorial plane of the cell revealed that the positive signals often appeared to be discontinuously distributed along the axopodia (
|
||
<figureCitation id="133B2A10675AFFFB125C8F4EFA13F801" box="[1377,1448,1919,1946]" captionStart="Fig" captionStartId="7.[125,160,682,704]" captionTargetBox="[125,1456,225,650]" captionTargetId="figure-7@7.[125,1456,225,651]" captionTargetPageId="7" captionText="Fig. 6. Confocal analysis of α-tubulin-immunolabeled extended axopodia. Fine reconstructed projection image from 161 optical sections taken at a 0.05 µm (top) and the corresponding light micrograph (bottom). Note the regions of relatively low fluorescence (arrowheads) did not correspond to the location of kinetocysts (arrows). Scale bar: 10 μm." figureDoi="http://doi.org/10.5281/zenodo.10994467" httpUri="https://zenodo.org/record/10994467/files/figure.png" pageId="4" pageNumber="5">Fig. 6</figureCitation>
|
||
). Following the induction of axopodial contraction, however, positive signals were detected within the completely contracted axopodia in the cell (
|
||
<figureCitation id="133B2A106759FFF815778BA9FD13FC29" box="[586,680,920,946]" captionStart="Fig" captionStartId="8.[132,167,1601,1623]" captionTargetBox="[132,1462,225,1562]" captionTargetId="figure-7@8.[132,1463,225,1563]" captionTargetPageId="8" captionText="Fig. 7. (A, B) The distribution of α-tubulin in the cell after rapid axopodial contraction. (A) Low-magnification images of the cell after the induction of rapid axopodial contraction. A projection image of the whole cell constructed from 87 optical sections taken at a 0.5 µm interval (left) and the corresponding light micrograph (right). (B) High-magnification images of the same cell shown in (A). A projection image of the equatorial plane of the cell body constructed from 5 optical sections taken at a 0.5 µm interval (left), and the corresponding light micrograph (right). Note that the positive signals in the distal parts of the axopodia have a branched appearance (arrowheads). Scale bars: 20 μm (A), 10 μm (B)." figureDoi="http://doi.org/10.5281/zenodo.10994469" httpUri="https://zenodo.org/record/10994469/files/figure.png" pageId="7" pageNumber="8">Fig. 7A</figureCitation>
|
||
). Those signals accumulated in the peripheral region of the cell (
|
||
<figureCitation id="133B2A106759FFF817B88BECFF5DFC6C" box="[133,230,989,1015]" captionStart="Fig" captionStartId="8.[132,167,1601,1623]" captionTargetBox="[132,1462,225,1562]" captionTargetId="figure-7@8.[132,1463,225,1563]" captionTargetPageId="8" captionText="Fig. 7. (A, B) The distribution of α-tubulin in the cell after rapid axopodial contraction. (A) Low-magnification images of the cell after the induction of rapid axopodial contraction. A projection image of the whole cell constructed from 87 optical sections taken at a 0.5 µm interval (left) and the corresponding light micrograph (right). (B) High-magnification images of the same cell shown in (A). A projection image of the equatorial plane of the cell body constructed from 5 optical sections taken at a 0.5 µm interval (left), and the corresponding light micrograph (right). Note that the positive signals in the distal parts of the axopodia have a branched appearance (arrowheads). Scale bars: 20 μm (A), 10 μm (B)." figureDoi="http://doi.org/10.5281/zenodo.10994469" httpUri="https://zenodo.org/record/10994469/files/figure.png" pageId="7" pageNumber="8">Fig. 7B</figureCitation>
|
||
). Moreover, the signals in the cell with contracted axopodia often exhibited a branched appearance in the distal part of axopodia (
|
||
<figureCitation id="133B2A106759FFF816E08C13FD82FBA7" box="[477,569,1058,1084]" captionStart="Fig" captionStartId="8.[132,167,1601,1623]" captionTargetBox="[132,1462,225,1562]" captionTargetId="figure-7@8.[132,1463,225,1563]" captionTargetPageId="8" captionText="Fig. 7. (A, B) The distribution of α-tubulin in the cell after rapid axopodial contraction. (A) Low-magnification images of the cell after the induction of rapid axopodial contraction. A projection image of the whole cell constructed from 87 optical sections taken at a 0.5 µm interval (left) and the corresponding light micrograph (right). (B) High-magnification images of the same cell shown in (A). A projection image of the equatorial plane of the cell body constructed from 5 optical sections taken at a 0.5 µm interval (left), and the corresponding light micrograph (right). Note that the positive signals in the distal parts of the axopodia have a branched appearance (arrowheads). Scale bars: 20 μm (A), 10 μm (B)." figureDoi="http://doi.org/10.5281/zenodo.10994469" httpUri="https://zenodo.org/record/10994469/files/figure.png" pageId="7" pageNumber="8">Fig. 7B</figureCitation>
|
||
,
|
||
<emphasis id="B974EA876759FFF815788C13FD6BFBA7" box="[581,720,1058,1084]" italics="true" pageId="7" pageNumber="8">arrowheads</emphasis>
|
||
).
|
||
</paragraph>
|
||
</subSubSection>
|
||
</treatment>
|
||
</document> |