The Cortical Division Zone

When a somatic plant cell prepares to divide, the cortical microtubular network disassembles and a ring, the preprophase band (PPB), consisting of microtubules (MTs) [FIG. 1A] and actin filaments is generated by altering the dynamics of the microtubule polymerization at the cortex [1,2]. This ring circumvents the usually centered nucleus and prophase spindle during prophase [3]. The PPB is removed before chromosome segregation and cytokinesis and is thought to predict the axis along which the cell is going to divide (the future division plane). As the position of the new cell wall after cytokinesis corresponds to the former position of the PPB, it was proposed that some factor should remain after PPB degradation to mark the division zone (i.e. the cortical area previously occupied by the PPB) throughout mitosis [4]. When the PPB MTs degrade, cortical actin disappears at the position of the PPB, leaving behind an actin-depleted zone (ADZ) [FIG. 1B] that lasts throughout mitosis [5].

The formation of the PPB and the ADZ provided the first molecular evidence for the establishment of a cortical division zone that acts as a signpost for the guidance of the expanding cell plate. Recently several other markers of the division zone were identified. A kinesin (KCA1) was found to translocate from the cytoplasm to the plasma membrane (PM) during PPB formation [FIG. 1C]. This kinesin marks the cortical division zone as it is specifically excluded from the PM at this zone [FIG.1 D, E], thus producing a KCA1 depleted zone (KDZ), analogous to the ADZ. The exclusion of KCA1 from the PM at the division zone indicates that next to the actin cytoskeleton, the PM itself is involved in the demarcation of this zone [6].

Figure 1 : Representative images of BY-2 cells during Cortical Division Zone Establishment.

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A. Projection of a preprophase band and forming prophase spindle highlighted using RFP-MAP4; B. Projection of a BY-2 cell expressing the Actin Binding Domain of Fimbrin fused to RFP showing the Actin Depleted Zone (ADZ, arrowheads); C. Confocal section of two BY-2 cells expressing GFP-KCA1 co-stained with the plasma membrane marker FM4-64 during interphase (right) and metaphase (left). KCA1 translocates from the cytoplasm to the plasma membrane during cell division while being specifically absent from the cortical division zone, thus forming the KCA1 Depleted Zone (KDZ); D. Projection of two BY-2 cells expressing GFP-KCA1 and RFP-TUA2. The upper cell shows the KDZ during metaphase while the lower cell shows the association of KCA1 to the plasma membrane during PPB formation; E. BY-2 cell expressing GFP-KCA1 stained with FM4-64 staining highlighting the absence of KCA1 from the cortical division zone during cytokinesis. (Fig. 1A courtesy of Daniel Van Damme; Fig. 1B,C,D,& E courtesy of Marleen Vanstraelen)

Next to the ADZ and the KDZ which are negative markers of the division zone, the first positive marker that lasts throughout mitosis and cytokinesis was also identified. The Arabidopsis TANGLED homologue (AtTAN) localizes to the PPB in a microtubule dependent manner, forming a broad ring that narrows down to a sharp and punctuate (beads on a string) ring after PPB breakdown and rapidly disassembles after cytokinesis [7]. Once the division zone is set, the plant cell can divide. Plant cytokinesis is initiated by de novo formation of a transient membrane compartment, the cell plate, at the spindle midzone by targeted vesicle transport and homotypic vesicle fusion. (Cell plate targeted probes is discussed by Sebastian Bednarek). The cell plate sandwiched between the two sets of parallel oriented MT bundles is referred to as the phragmoplast [3], which expands centrifugally toward the cell periphery [8]. The two sets of microtubules of the phragmoplast are stabilized by microtubule-bundling proteins like the members of the MAP65 family. One member of this family, AtMAP65-3/PLEIADE is part of a stabilization mechanism anchoring the two sets of MTs as this protein localizes at the centrally located plus ends of the microtubules [9,10] [FIG. 2A] and mutants show cytokinesis defects associated with widened phragmoplasts [9]. The phragmoplast MTs translocate laterally by polymerization of microtubules at the outer rim and depolymerization of microtubules at the inner side, causing the initial disc-shaped phragmoplast to transform to a donut-like phragmoplast. The process of lateral expansion is driven by a Mitogen Activated protein kinase (AtNACK-MAPK) pathway which in BY-2 cells leads to the phosphorylation and down-regulation of the microtubule stabilizing properties of NtMAP65-1 [11].

Figure 2 : Phragmoplast microtubules and the forming cell plate

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A. Projection of a BY-2 cell expressing RFP-MAP4 and AtMAP65-3-GFP during cell plate formation. RFP-MAP4 labels the phragmoplast microtubules along the entire length while AtMAP65-3 is concentrated at the inner (plus) ends.

B-C. Dividing Arabidopsis root cells expressing TPLATE-GFP. TPLATE localizes to the forming cell plate and to the plasma membrane at the cortical division zone during cell plate anchoring (yellow arrowheads). (Figures courtesy of Daniel Van Damme)

The expanding phragmoplast is guided by an unknown mechanism that besides AtTAN involves two kinesins, POK1 and POK2 [12] until it reaches the cortical area previously occupied by the PPB. The last phase of cytokinesis consists of the fusion of the cell plate with the plasma membrane and the maturation of the callose-rich cell plate to a cellulosic cell wall. The mechanism involved in this heterotypic fusion step between the PM and the cell plate involves the TPLATE protein as knockout or downregulation of this gene causes problems in anchoring the cell plate to the correct insertion site in Arabidopsis and in BY-2 cells. The TPLATE protein localizes to the forming cell plate and marks the PM at the division zone during cell plate insertion [FIG.2 B,C]. TPLATE contains domains with similarity to adaptin and coat proteins, indicating that it is involved in membrane trafficking events [10]. AIR9 is another protein with a likely role in cell plate maturation. When constitutively expressed in BY-2 cells, GFP-AIR9 associates with the cortical microtubular cytoskeleton and the PPB, in agreement with a MT-binding domain at the N-terminus of the polypeptide. During cell plate fusion with the PM, AIR9-GFP is specifically targeted to the exact insertion site and later on spreads into the formed cell plate, forming a torus. The accumulation of AIR9 at the insertion site occurs before the reformation of the cortical microtubular network, indicating that this protein is likely involved in the maturation of the cell plate and/or the initiation of the cortical microtubular network [13].

Fluorescent Probes created for visualizing the establishment of Cortical Division Zone

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