Journal: bioRxiv
Article Title: Plasma membrane and cytoplasmic compartmentalization: a dynamic structural framework required for pollen tube tip growth
doi: 10.1101/2024.04.08.588543
Figure Lengend Snippet: A) Alignment of linear bars representing all PM domains associated with various proteins and lipids ( , and ), or in contact with different cytoplasmic structures or regions , which were investigated in this study. Mean meridional distances (MDs) from the pollen tube apex (MD = 0 µm) of the proximal (if applicable) and distal endpoints of these PM domains were quantitatively determined (values indicated next to each bar and in ) and statistically compared using one-way ANOVA with Tukey Kramer (HSD) testing. Error bars: standard deviation. Distinct letters indicate statistically significantly different mean MDs from the apex (p < 0.05). Red vertical lines: maximal RFI indicated in , , and . Number of pollen tubes/domain endpoints analyzed (both sides of pollen tubes were analyzed, if possible): PI4P, n = 70/140; PI4,5P 2 , n = 83/155; DAG, n = 65/130; PS, n = 60/119; PA, n = 81/162; NtPLC3, n = 117/231; NtPI4P-5K4, n = 55/106; NtRAC5, n = 55/107; RAC/ROP GTP , n = 66/132; NtGEF12a, n = 61/122; NtGAP1, n = 67/134; F-actin fringe, n = 63/126; TGN, n = 69/138; VAR, n = 57/114; Ca 2+ , n = 67/134. For direct comparison, the extension of the apical PM domain displaying high membrane order as well as the position of the lateral peak in membrane order are also indicated . The apical PM domain displaying high membrane order is identical to the secretory domain. Light blue shading indicates a PM region in which the endpoints of many apical and lateral domains are positioned. All domains are drawn to scale. B) Left column: medial confocal optical sections through representative pollen tubes stably expressing the indicated fluorescent fusion proteins serving as markers for the following cytoplasmic structures or regions: F-actin (LIFEACT-eYFP), subapical TGN compartment (eYFP-RISAP), VAR (NtINT4-eYFP) or region displaying elevated Ca 2+ concentrations (2xR-GECO1). Numbers (top right) indicate the growth rate (µm/min) of individual pollen tubes shown, which was determined after image acquisition (average growth rate of all analyzed pollen tubes: Supplemental Fig. S1). Scale bars: 8 µm. Right column: quantitative analysis of PM-associated fluorescence (directly underneath the PM) in all analyzed pollen tubes expressing LIFEACT-eYFP (n = 59 pollen tubes, 110 RFI distribution patterns), eYFP-NtRISAP (n = 58 pollen tubes, 116 RFI distribution patterns), NtINT4-eYFP (n = 55 pollen tubes, 109 RFI distribution patterns) or 2xR-GECO1 (n = 55 pollen tubes, 108 RFI distribution patterns). Both sides of imaged pollen tubes were analyzed, if possible. Light green dots: mean relative fluorescence intensity (RFI) associated with the PM at different meridional distances (MD) from the apex (MD = 0 µm). Dark grey vertical lines: standard deviation. Red vertical lines: maximal RFI. Black vertical lines delimiting light grey shading: proximal (if applicable) and distal half-maximal RFI.
Article Snippet: The haploid yeast strains THY.AP4 and THY.AP5 ( ) were transformed with pMetOYC-NtGEF12a or pNubG-NtRAC5, respectively, and plated on appropriate selective media (CSM +Ade, His, Trp, Ura or CSM +Ade, His, Leu; MP Biomedicals) according to .
Techniques: Standard Deviation, Comparison, Membrane, Stable Transfection, Expressing, Fluorescence
![Left column: medial confocal optical sections through representative pollen tubes expressing the indicated eYFP fusion proteins, or free eYFP used as control, either transiently (CRIB NtGAP1 -eYFP) or stably (all other fluorescent proteins). Numbers (top right) indicate the growth rate (µm/min) of the individual pollen tubes shown, which was determined after image acquisition (average growth rate of all analyzed pollen tubes: Supplemental Fig. S1). Scale bars: 8 µm. Right column: quantitative analysis of PM-associated fluorescence (directly underneath [eYFP] or at the PM) in all analyzed pollen tubes expressing each indicated protein. Light green dots: mean relative fluorescence intensity (RFI) associated with the PM at different meridional distances (MDs) from the apex (MD = 0 µm). Both sides of imaged pollen tubes were analyzed, if possible. Dark grey vertical lines: standard deviation. Red vertical lines: maximal RFI. Black vertical lines delimiting light grey shading: proximal and distal half-maximal RFI (if applicable). A) Intracellular distribution and PM association of <t>eYFP-NtRAC5</t> (n = 49 pollen tubes, 95 RFI distribution patterns), along with the intracellular distribution of free eYFP (n = 55 pollen tubes, 110 RFI distribution patterns). B) Intracellular distribution and PM association of CRIB NtGAP1 -eYFP serving as a marker for GTP-bound active RAC/ROP GTP (n = 61 pollen tubes, 118 RFI distribution patterns), as well as of eYFP fused to NtGEF12a (n = 55 pollen tubes, 110 RFI distribution patterns) or to NtGAP1 (n = 65 pollen tubes, 128 RFI distribution patterns), which activate or inactivate RAC/ROP signaling functions, respectively.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_43/10__1101_slash_2024__04__08__588543/10__1101_slash_2024__04__08__588543___F2.large.jpg)
