Glycolysis regulates pollen tube polarity via Rho GTPase signaling

<div><p>As a universal energy generation pathway utilizing carbon metabolism, glycolysis plays an important housekeeping role in all organisms. Pollen tubes expand rapidly via a mechanism of polarized growth, known as tip growth, to deliver sperm for fertilization. Here, we report a novel and surprising role of glycolysis in the regulation of growth polarity in <i>Arabidopsis</i> pollen tubes via impingement of Rho GTPase-dependent signaling. We identified a <i>cytosolic phosphoglycerate kinase</i> (<i>pgkc-1</i>) mutant with accelerated pollen germination and compromised pollen tube growth polarity. <i>pgkc-1</i> mutation greatly diminished apical exocytic vesicular distribution of REN1 RopGAP (Rop GTPase activating protein), leading to ROP1 hyper-activation at the apical plasma membrane. Consequently, <i>pgkc-1</i> pollen tubes contained higher amounts of exocytic vesicles and actin microfilaments in the apical region, and showed reduced sensitivity to Brefeldin A and Latrunculin B, respectively. While inhibition of mitochondrial respiration could not explain the <i>pgkc-1</i> phenotype, the glycolytic activity is indeed required for PGKc function in pollen tubes. Moreover, the <i>pgkc-1</i> pollen tube phenotype was mimicked by the inhibition of another glycolytic enzyme. These findings highlight an unconventional regulatory function for a housekeeping metabolic pathway in the spatial control of a fundamental cellular process.</p></div

Vesicle trafficking in the <i>pgkc-1</i> mutant.

<p><b>(A)</b> EYFP-RABA4D signal in WT and <i>pgkc-1</i> pollen tubes subjected to mock or 0.4 μM BFA treatment. Scale bar = 5 μm. <b>(B)</b> EYFP-RABA4D signal intensity. Measurements were performed as described in Materials and Methods. Fifteen to twenty pollen tubes from each sample were measured. 0 μm indicates the position of apical tip. Error bars on curves indicate standard error. <b>(C and D)</b> WT and <i>pgkc-1</i> pollen tube morphology when subjected to <b>(C)</b> mock and <b>(D)</b> 0.4 μM BFA treatment. Scale bar = 50 μm. <b>(E to G)</b> WT and <i>pgkc-1</i> plant pollen germination <b>(E)</b>, pollen tube length <b>(F)</b>, and pollen tube width <b>(G)</b> when subjected to mock or 0.4 μM BFA treatment. Bars represent mean ± SEM. Asterisks indicate significant differences versus mock treatment as determined using Student’s <i>t</i>-test (** = p < 0.001).</p

<i>gapcp1/gapcp2</i> double mutant is also defective in pollen tube polarity.

<p><b>(A)</b> WT pollen tubes. <b>(B)</b> <i>gapcp1/gapcp2</i> double mutant pollen tubes. Scale bar = 50μm. <b>(C)</b> Average length of pollen tubes. <b>(D)</b> Average width of pollen tubes. Bars represent mean ± SEM. Asterisks indicate significant differences versus WT as determined using Student’s <i>t</i>-test (** = p < 0.001).</p

<i>pgkc-1</i> mutant exhibits enhanced pollen germination and growth depolarization.

<p><b>(A)</b> WT pollen tube morphology. <b>(B)</b> <i>pgkc-1</i> (SALK_066422C) pollen tube morphology. <b>(C)</b> Complemented <i>pgkc-1</i> pollen tube morphology. Scale bar = 50 μm. <b>(D)</b> Pollen germination rate at 3 h and 9 h, respectively. <i>pgkc-1</i> germinated at higher rates than WT and complemented pollen, especially at the early time point. (<b>E</b>) Pollen tube length of WT, <i>pgkc-1</i> mutant, and genetically complemented <i>pgkc-1</i> plants at 9 h after germination. <b>(F)</b> Pollen tube width of WT, <i>pgkc-1</i> mutant, and genetically complemented <i>pgkc-1</i> plants at 9 h after germination. Bars represent mean ± SEM. Asterisks indicate significant differences (** = p < 0.001) versus WT as determined by Student’s <i>t</i>-test.</p

ROP1 signaling in the <i>pgkc-1</i> mutant.

<p><b>(A)</b> Active ROP1 visualized by CRIB4-GFP signal in WT and <i>pgkc-1</i> pollen tubes. Scale bar = 5μm. <b>(B)</b> Average CRIB4-GFP signal intensity along WT and <i>pgkc-1</i> pollen tubes. <b>(C)</b> GFP-REN1 localization in WT and <i>pgkc-1</i> pollen tubes. Scale bar = 5 μm. <b>(D)</b> Average GFP-REN1 signal intensity along WT and <i>pgkc-1</i> pollen tubes. Measurements were performed as described in Materials and Methods. Fifteen pollen tubes from each sample were measured. The 0 μm label indicates the position of the apical tip. Error bars on curves indicate standard error of the mean. <b>(E to G)</b> Pollen tube morphology of <i>pgkc-1</i> <b>(E)</b>, <i>ren1-3</i> <b>(F)</b>, and <i>ren1-3/pgkc-1</i> double mutant plants <b>(G)</b>. Scale bar = 50 μm. <b>(H and I)</b> Pollen tube length <b>(H)</b> and width <b>I)</b> of WT, <i>pgkc-1</i>, <i>ren1-3</i>, and <i>ren1-3/pgkc-1</i> double mutant plants. Bars represent mean ± SEM. Asterisks indicate significant differences versus single mutant plant as determined using Student’s <i>t</i>-test (** = p < 0.001).</p

Effects of disrupting GAPDH on pollen tube polarity.

<p><b>(A)</b> Glycolytic pathway of GAPDH and PGK. <b>(B to G)</b> Pollen tube morphology of WT <b>(B)</b>, <i>pgkc-1</i> <b>(C)</b>, and <i>ren1-3</i> <b>(D)</b> plants subjected to mock treatment; pollen tube morphology of WT <b>(E)</b>, <i>pgkc-1</i> <b>(F)</b>, and <i>ren1-3</i> <b>(G)</b> plants treated with 40 μM CGP 3466B. Both <i>pgkc-1</i> and <i>ren1-3</i> plants were dramatically depolarized by CGP medium. Scale bar = 50 μm. <b>(H and I)</b> Pollen tube length <b>(H)</b> and width <b>(I)</b> of WT, <i>pgkc-1</i>, and <i>ren1-3</i> pollen tubes subjected to mock and 40 μM CGP treatment. Bars represent mean ± SEM. Asterisks indicate significant differences versus either single mutant as determined using Student’s <i>t</i>-test with either single mutant (** = p < 0.001). <b>(J)-(M)</b> Average signal intensity along WT pollen tubes subjected to mock or CGP treatment of <b>(J)</b> GFP- REN1, <b>(K)</b> CRIB4-GFP, <b>(L)</b> Lifeact-mEGFP, <b>(M)</b> EYFP-RABA4D. Measurements were performed as described in Materials and Methods. Fifteen pollen tubes from each sample were measured. The 0 μm label indicates the position of the apical tip.</p

F-actin dynamics in the <i>pgkc-1</i> mutant.

<p><b>(A)</b> Lifeact-mEGFP signal in WT and <i>pgkc-1</i> pollen tubes with mock or 1.5 nM LatB treatment. Scale bar = 5 μm. <b>(B)</b> Average GFP signal intensity along WT and <i>pgkc-1</i> pollen tubes with mock or 1.5 nM LatB treatment. Measurements were performed as described in Materials and Methods. Thirty-five pollen tubes were measured for each sample. The 0 μm indicates the position of the extreme tip. Orange line indicates WT pollen tube; red line indicates WT pollen tube treated with 1.5 nM LatB; gray line indicates <i>pgkc-1</i> pollen tube; black line indicates <i>pgkc-1</i> pollen tube treated with 1.5 nM LatB. Error bars on curves indicate standard error of the mean. <b>(C and D)</b> WT and <i>pgkc-1</i> pollen tube growth when subjected to mock medium <b>(C)</b> or 1.5 nM LatB <b>(D)</b> treatment. Scale bar = 50 μm. <b>(E to G)</b> WT and <i>pgkc-1</i> plant pollen germination <b>(E)</b>, pollen tube length <b>(F)</b>, and pollen tube width <b>(G)</b> when subjected to mock or 1.5 nM LatB treatment. Bars represent mean ± SEM. Asterisks indicate significant differences versus mock treatment as determined using Student’s <i>t</i>-test (** = p < 0.001).</p

Proposed model for potential role of glycolysis in pollen tube polarity.

<p>Glycolysis is required for the association of the REN1 RopGAP with exocytic vesicles. REN1 RopGAP negatively regulates the Rho GTPase signaling, which coordinates pollen tube growth by coordinating actin dynamics and exocytosis. The mechanisms linking glycolysis with cell polarity remains elusive, which are possibly energization of unclear vesicle trafficking, or HXK signaling.</p

Catalytically inactive mPGKc could not rescue the <i>pgkc-1</i> mutant phenotype.

<p><b>(A)</b> Protein sequences of the conserved Glutamate179 regions of PGK proteins. AtPGKc, <i>Arabidopsis thaliana</i> cytosolic PGK; OsPGKc, <i>Oryza sativa</i> cytosolic PGK; DmPGK, <i>Drosophila melanogaster</i> PGK; ScPGK, <i>Saccharomyces cerevisiae</i> PGK; EcPGK, <i>Escherichia coli</i> PGK. Glutamate179 in AtPGKc is labeled with a red dot. <b>(B)</b> <i>PGKc</i> expression level. <b>(C)</b> Pollen germination rate. <b>(D)</b> Average length of pollen tubes. <b>(E)</b> Average width of pollen tubes. Bars represent mean ± SEM. Asterisks indicate significant differences versus WT as determined using Student’s <i>t</i>-test (** = p < 0.001).</p

Chinese expert consensus on cone‐beam CT‐guided diagnosis, localization and treatment for pulmonary nodules

Abstract Cone‐beam computed tomography (CBCT) system can provide real‐time 3D images and fluoroscopy images of the region of interest during the operation. Some systems can even offer augmented fluoroscopy and puncture guidance. The use of CBCT for interventional pulmonary procedures has grown significantly in recent years, and numerous clinical studies have confirmed the technology's efficacy and safety in the diagnosis, localization, and treatment of pulmonary nodules. In order to optimize and standardize the technical specifications of CBCT and guide its application in clinical practice, the consensus statement has been organized and written in a collaborative effort by the Professional Committee on Interventional Pulmonology of China Association for Promotion of Health Science and Technology