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Comparative Study
, 3, 11

Microtubule Plus-Ends Reveal Essential Links Between Intracellular Polarization and Localized Modulation of Endocytosis During Division-Plane Establishment in Plant Cells

Affiliations
Comparative Study

Microtubule Plus-Ends Reveal Essential Links Between Intracellular Polarization and Localized Modulation of Endocytosis During Division-Plane Establishment in Plant Cells

Pankaj Dhonukshe et al. BMC Biol.

Abstract

Background: A key event in plant morphogenesis is the establishment of a division plane. A plant-specific microtubular preprophase band (PPB) accurately predicts the line of cell division, whereas the phragmoplast, another plant-specific array, executes cell division by maintaining this predicted line. Although establishment of these specific arrays apparently involves intracellular repolarization events that focus cellular resources to a division site, it still remains unclear how microtubules position the cell division planes. Here we study GFP-AtEB1 decorated microtubule plus-ends to dissect events at the division plane.

Results: Early mitotic events included guided growth of endoplasmic microtubules (EMTs) towards the PPB site and their coincident localization with endocytic vesicles. Consequently, an endosomal belt lay in close proximity to the microtubular PPB at its maturation and was maintained during spindle formation. During cytokinesis, EMTs radiated from the former spindle poles in a geometrical conformation correlating with cell-plate navigation and tilt-correction. Naphthylphtalamic acid (NPA), an inhibitor of polar auxin efflux, caused abnormal PPBs and shifted division planes.

Conclusion: Our observations reveal a spatio-temporal link between microtubules and intracellular polarization essential for localized endocytosis and precise establishment of the division plane in plants. Additionally, they implicate the growth regulator, auxin, in this important cellular event.

Figures

Figure 1
Figure 1
Mechanisms for microtubule guidance and bundling. Green: GFP-AtEB1 (in A-H and N) and GFP-MAP4 (in I-M). Red: YFP-MAP4 (in C, E-G and N) and YFP-CLIP170 (in D). (A) 3-D maximum projection of interphase BY-2 cell, highlighting punctuate GFP-AtEB1 labeling in the cortex. (B) In interphase cells, GFP-AtEB1 comets at the cortex move in the same or opposite direction on the same track (arrowheads), and sometimes together (arrow) (See additional file 1: Movie 1). (C) In co-transformed cells, GFP-EB1 labels the growing ends of microtubules labeled by YFP-MAP4 (See additional file 2: Movie 2). (D) Co-localization of GFP-AtEB1 and YFP-CLIP170 on growing microtubule plus ends (See additional file 3: Movie 3). (E) GFP-AtEB1 labeled growing microtubule changing from one microtubule track (labeled with YFP-MAP4) to another. [58] Growth of two separate unbundled microtubules (arrowheads) transiently meeting (arrow) and afterwards separating without inducing catastrophe. (G) Microtubules growing in opposite directions on the same track with similar speed (arrowheads) meet (arrow) and continue growing in opposite directions without inducing catastrophe. (H) Microtubule nucleation and growth (arrowheads) on an already existing microtubule (arrow). (I) Situation where attachment of a microtubule plus end (yellow arrowhead) to an existing microtubule induces a translocation of the minus end (red arrowhead) from one microtubule to another. (J) Microtubule plus end (yellow arrowhead) growing towards an existing microtubule, followed by guided growth in a new direction, causes bending at the point of previous attachment (red arrowhead). (K) Treadmilling microtubule (red arrowhead) with its plus end (red arrow) and minus end (yellow arrowhead) moving in the same direction (yellow arrowheads and red arrows) initiates bundling by bridging two other separate preexisting microtubules. (L) Long microtubule growing (arrowhead) and interacting with an existing microtubule induces reorientation of growth and bending, resulting in bundling with a shorter growing microtubule. (M) Depolymerization of one microtubule partially associated with a bundle (yellow arrowhead) causes bending of the remaining structure (red arrowhead). (N) Microtubule minus end detachment and subsequent movement (arrowheads) induces loss of GFP-AtEB1 from its plus end (yellow arrows); it recovers GFP-AtEB1 labeling and plus end growth once its minus end acquires new support on another polymer (arrowhead). Note that the same microtubule bends (red arrow) when its minus end is fixed and the plus end (yellow arrow) hits another microtubule. Time is indicated in seconds and bars represent 5 μm in A-D, 3 μm in K-M, 2 μm in E, I, J and 1 μm in F-H, N.
Figure 2
Figure 2
Polarity and growth speed of EMTs bridging nucleus and cortex. Green: GFP-AtEB1 (in A-L). (A-F) EMTs exhibit bidirectional growth and microtubule bundling. Note that that the microtubule originating from the nuclear surface (outgoing) and the one coming from the cortex (incoming) cross each other (arrow) and, as in the cortical array, grow with similar speeds without interfering each other (arrowheads) (see additional file 4: Movie 4). (G-L) EMT plus ends radiating mainly in an outward direction from the NE during PPB maturation (see additional file 5: Movie 5). Kymograph projection of microtubule plus ends in the interphase cortex (M), PPB cortex (N) and preprophase cytoplasm (O) showing sustained polymerization. The horizontal axis, d, represents distance (18 μm in M, 13 μm in N and 20 μm in O), and the vertical axis, t, represents time (290 s in M, 140 s in N and 390 s in O). Note that for each of the 3 cases (M-O), the microtubules follow the tracks, exhibit bi-directionality and grow with the same speeds. By comparing the slopes between images M-O, it becomes evident that the microtubule growth speed increases from interphase to the PPB stage, as previously reported [9]. Note that the arrowhead in M shows the crossing of two EMTs growing on the same path at the same time but in opposite directions. Nucleus is marked by 'N', time is indicated in seconds and bars represent 8 μm.
Figure 3
Figure 3
EMTs configure the premitotic cytoplasm. Green: GFP-MAP4 (in A-I). Red: FM4-64 (in A, E and F), Alexa 633 (in B), ST-YFP (in C), Mitotracker (in D) FM4-64 labeled endosomes (A), Alexa 633 labeled pinocytic vesicles (B), ST-YFP labeled GA (C) and Mitotracker labeled mitochondria (D) all remain in the vicinity of GFP-MAP4 marked EMT tracks. (E) At interphase, GFP-MAP4 labeled microtubules remain in the cortex and FM4-64 labeled vacuoles occupy most of the endoplasmic space. [F] At preprophase, GFP-MAP4 labeled EMTs intersect the vacuoles labeled by FM4-64. Premitotic cells treated with latrunculin B (G), oryzalin (H) or both (I) show cytoplasmic disorganization in the presence of oryzalin (H-I). Bars represent 8 μm.
Figure 4
Figure 4
Endosomal belt co-localizes with microtubular PPB during preprophase. Green: GFP-MAP4 (in A, B, L, M and P), GFP-AtEB1 (in C-K), GFP-Ara7 (in N) and ST-YFP (in O). Red: FM4-64 (in A-M and O-P). Early in the G2-M transition, FM4-64 labeled endocytic vesicles follow the emerging EMTs labeled with GFP-MAP4, as shown in single median section (A) and 3D-projection (B). The marked rectangle in (C) is zoomed in for (D-I). FM4-64 labeled endocytic vesicles preferentially internalize from the cortical areas approached by the GFP-AtEB1 labeled EMT plus ends (D-F) (see additional file 6: Movie 6), and oryzalin-induced microtubule depolymerization disrupts their internalization routes (G-I) (see additional file 7: Movie 7) whereas the internalization paths are recovered after oryzalin removal (J). (K) Close-up of GFP-AtEB1 marked EMT plus ends bridging the NE and PPB. Note that during PPB narrowing, FM4-64 labeled endocytic vesicles preferentially internalize from the cortical areas approached by the GFP-AtEB1 (see additional file 8: Movie 8). Formation of an FM4-64 labeled cortical belt at the PPB site (labeled with GFP-MAP4) is shown in a single median section (L) and in 3-D projection (M). (N) 3-D projection of GFP-Ara7 labeled endosomes exhibiting an endosomal belt at preprophase. (O) Both FM4-64 labeled endosomes and ST-YFP labeled GAs form a cortical belt at the PPB site. (P) GFP-MAP4 labeled EMTs connecting the nucleus to the PPB intersect FM4-64 labeled vacuoles. Time is indicated in minutes. Bars represent 7 μm in A, B, J, L, M, 10 μm in C, N-P and 5 μm in K.
Figure 5
Figure 5
PM targeted EMT plus ends probe the areas occupied by the preceding PPB and align the cell plates for proper docking at the parental walls. Green: GFP-MAP4 (in A, F-I), GFP-AtEB1 (in B-E, J-O and P). Red: FM4-64 (in A, F-O), YFP-MAP4 (in P). (A) Discontinuity of the vacuolar structures in the preceding PPB site (arrowheads) is maintained at the spindle stage, as visualized with FM4-64 labeled vacuoles and GFP-MAP4 labeled microtubules.(B-C) At the onset of the phragmoplast stage, GFP-AtEB1 labeled EMT plus ends (red arrowheads) originating from the former spindle poles grow towards the cortex (see additional file 9: Movie 9). Occasionally, they grow towards the polar areas (yellow arrowhead). (D) GFP-AtEB1 labeled EMT plus ends (arrowheads) are attracted to the cortical areas marked by the preceding PPB. At late telophase, the distance through which GFP-AtEB1 labeled EMT plus ends reach towards the cortex is reduced. (E) 3-D projection showing GFP-AtEB1 labeled EMT plus end trajectories directed towards the cortex, which are different from the main phragmoplast structure. GFP-MAP4 labeled EMTs (F-I) or GFP-AtEB1 labeled EMT plus ends (J-M) continue to reach the cortex at the former PPB site and display close proximity to FM4-64 labeled endosomes (red arrow and arrowheads). These endosomes display movement towards the minus end of these EMTs. (N-O) GFP-AtEB1 labeled plus end growth of EMTs (arrowheads) towards opposite sides of the cortex is maintained during cell plate and phragmoplast tilting (see additional file 10: Movie 10). (P) Enrichment of GFP-AtEB1 labeled microtubule plus ends (arrowhead) but not of YFP-MAP4 labeled microtubular parts at the phragmoplast midline. Time in F-I is given in seconds while that in J-O is indicated in minutes. Bars in A-O represent 8 μm while that in P represents 10 μm.
Figure 6
Figure 6
NPA induces abnormal PPBs and altered cell divisions. (A-B) Normal anticlinal cell divisions (arrowheads in A) and transverse organization of cortical microtubules in NPA untreated cells. (C-D) Inclined and periclinal cell divisions (red arrowheads in C) with altered organization of GFP-MAP4 labeled cortical microtubules (red arrowheads in D) in NPA treated cells. A, C show single median sections and B, D show 3-D projections. Note that the first round of cell division (yellow arrowheads) is normal and a shift in the cell division planes occurs in the second round. (E-F) Formation of periclinal PPBs and spindles (arrowheads in E) and periclinal phragmoplasts (arrowhead in F). Bidirectional arrows in A, C, E and F show the long axes of the cells. (G-L) NPA treatment sometimes causes formation of two separate PPBs (arrowheads in G) equidistant from the nucleus, which results in tilted spindle formation (I) and phragmoplast initiation (J), phragmoplast growth (K) and cell plate docking (L) at sites marked by either of the PPBs (arrowheads) (see additional file 11: Movie 11). G shows 3-D projection and H-L show single median sections. Bars represent 10 μm and time is indicated in minutes.

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