PIN2 Polarity Establishment in Arabidopsis in the Absence of an Intact Cytoskeleton

Cell polarity is crucial for the coordinated development of all multicellular organisms. In plants, this is exemplified by the PIN-FORMED (PIN) efflux carriers of the phytohormone auxin: The polar subcellular localization of the PINs is instructive to the directional intercellular auxin transport, and thus to a plethora of auxin-regulated growth and developmental processes. Despite its importance, the regulation of PIN polar subcellular localization remains poorly understood. Here, we have employed advanced live-cell imaging techniques to study the roles of microtubules and actin microfilaments in the establishment of apical polar localization of PIN2 in the epidermis of the Arabidopsis root meristem. We report that apical PIN2 polarity requires neither intact actin microfilaments nor microtubules, suggesting that the primary spatial cue for polar PIN distribution is likely independent of cytoskeleton-guided endomembrane trafficking.


Introduction
Auxin is arguably the single most important regulator of plant growth and development. Pivotal to its function are auxin distribution gradients, or local asymmetries in the levels of auxin signaling between cells. The auxin gradients, besides requiring local auxin biosynthesis, depend largely on polarized intercellular auxin transport, which is in turn mediated by multiple families of auxin transporters [1][2][3]. The directionality of auxin flow is determined by the polar subcellular localization of the PIN-FORMED (PIN) auxin efflux carriers [4]. The regulation of cell polarity in general, and PIN polarity in particular, is thus a crucial, yet still mostly unanswered, question in plant biology. PIN polar localization is thought to be regulated mainly at the level of PIN endomembrane trafficking [5]. The movement of endomembrane vesicles depends on the cytoskeleton, and accordingly, pharmacological interference with actin or microtubules (MTs) has been linked to defects in PIN subcellular dynamics [6,7]. However, opinions on the importance of the cytoskeleton for PIN polarity as such differ [7,8]. Here, we analyzed the effects of cytoskeleton-depolymerizing agents on the generation of apical PIN2 polarity in Arabidopsis roots using time-lapse imaging of advanced fluorescent reporters. We found, surprisingly, that the correctly polarized subcellular PIN distribution can be achieved in the absence of both intact actin and MTs. Our findings suggest that the primary PIN polarization mechanism is likely independent of cytoskeleton-mediated endomembrane trafficking.

Results
During cytokinesis of Arabidopsis root cells, PIN polarity is transiently lost and subsequently re-established [6,[8][9][10]. We have recently shown that, in combination with the temporally-restricted expression of PIN2-GFP from the cytokinesis-specific KNOLLE promoter, the dividing cells can be used as a model to study PIN polarity establishment in real time [10]. Here, we employed this system together with the actin-and MT-depolymerizing drugs Latrunculin B (LatB) and Oryzalin (Ory), respectively, to test the importance of the cytoskeleton for the establishment of PIN polarity.
Application of either of the two drugs caused severe cytokinetic defects, ranging from division plane misorientation to complete cell plate (CP)-formation failure, which are consistent with both actin and MTs playing key roles in multiple steps of cell division [11]. This provided a good internal control which confirmed that the treatments indeed disrupted the respective cytoskeletal network (Figure 1a-c, Video S1). In both cases, the KNOLLE::PIN2-GFP signal was partly mislocalized to abnormal endomembrane compartments, providing further evidence of the functionality of the drugs. In spite of the disturbed cytoskeleton, however, apical polarity of the plasma membrane (PM)-localized signal was in both cases eventually achieved, albeit with a considerable delay (Figure 1b,c,g, Video S1). Notably, in case of the Ory treatment, the establishment of apical polarity was preceded by apolar distribution of the PM signal ( Figure 1c, Video S1).
We next applied both LatB and Ory at the same time. This co-treatment led to efficient depolymerization of both actin and MTs, as visualized by the respective markers Fimbrin-GFP and GFP-MAP4 (Figure 1e-f), and completely abolished the cells' ability to divide (Figure 1d, Video S1). Under these conditions, much of KNOLLE::PIN2-GFP localized to ectopic endomembrane aggregates that displayed only slow random movement and never reached the PM, indicating that PIN2-GFP trafficking was indeed severely disrupted ( Figure 1d, Video S1). Nevertheless, some of the signal did appear at the PM, presumably due to fusion events between the PM and vesicles that appeared in its proximity by Brownian motion. This PM signal was initially apolar, as in the case of the Ory treatment alone. Nonetheless, surprisingly, apical polarity of the PM-bound signal was eventually correctly established in most cases, albeit significantly later compared to the mock-treated control or either of the treatments alone (Figure 1d,g, Videos S1 and S2). This shows that the cellular mechanism that establishes apical PIN2 apical polarity is independent of the intact cytoskeletal network.
To validate our findings outside of the context of (unsuccessfully) dividing cells, we generated an RPS5a::LOX:nls-mCherry:lox::PIN2-GFP construct and introduced it into the HS::CRE background. In the resulting plants, a 1-hour heat shock led to the onset of Cre/Lox recombination-mediated expression of PIN2-GFP only in a few random and therefore often isolated cells, thus allowing us to observe the dynamic generation of PIN2 polarity in interphase cells as well (Figure 2a,b). Consistently with the previous results, the LatB + Ory cotreatment did not prevent apical PIN2-GFP polarity generation in this experiment either (Figure 2c).
the Ory treatment alone. Nonetheless, surprisingly, apical polarity of the PM-bound signal was eventually correctly established in most cases, albeit significantly later compared to the mock-treated control or either of the treatments alone (Figure 1d,g, Video S1, S2). This shows that the cellular mechanism that establishes apical PIN2 apical polarity is independent of the intact cytoskeletal network.  an RPS5a::LOX:nls-mCherry:lox::PIN2-GFP construct and introduced it into the HS::CRE background. In the resulting plants, a 1-hour heat shock led to the onset of Cre/Lox recombination-mediated expression of PIN2-GFP only in a few random and therefore often isolated cells, thus allowing us to observe the dynamic generation of PIN2 polarity in interphase cells as well (Figure 2a,b). Consistently with the previous results, the LatB + Ory cotreatment did not prevent apical PIN2-GFP polarity generation in this experiment either (Figure 2c).

Discussion
We have shown that that the application of cytoskeleton-interfering drugs Latrunculin B, Oryzalin, or both at the same time did not prevent the establishment of apical polarity of PIN2 in

Discussion
We have shown that that the application of cytoskeleton-interfering drugs Latrunculin B, Oryzalin, or both at the same time did not prevent the establishment of apical polarity of PIN2 in newly divided or interphase cells. The defects of the root growth rate, endomembrane trafficking, and cytokinesis caused by the drugs were consistent throughout the experiments, suggesting that the effects of the drugs were stable (Video S1 and data not shown). In all experiments presented, depolymerization of actin, MTs, or both at the same time led to partial PIN2-GFP mislocalization to abnormal endomembrane compartments. This again confirms that both cytoskeletal components are involved in PIN intracellular trafficking, as previously proposed [6][7][8]. On the other hand, the transient apolar PIN2-GFP localization preceded polarity establishment in LatB + Ory-treated cells only when expressed from the KNOLLE promoter, suggesting that it reflected the specific situation at the G2/M phase of the cell cycle and not the general behavior of PIN2. Moreover, the polar distribution of KNOLLE::PIN2-GFP appeared later under all treatments, demonstrating that cytoskeleton-guided trafficking contributes to the speed and/or efficiency of PIN polar targeting. Nevertheless, the spatial cue for PIN2-GFP polarity establishment was cytoskeleton-independent in both mitotic and interphase cells, supporting similar conclusions drawn in the past for PIN1 [8]. This suggests that actin-and MT-guided endomembrane trafficking stabilizes cell polarity by reinforcing asymmetrical distribution of polar cargoes, including the PINs. However, both actin and MTs act downstream of an unknown, cytoskeleton-independent polarity establishment mechanism.
Therefore, cytoskeleton-dependent polarized exocytosis, either secretory or recycling, cannot be the main mechanism of PIN polarity generation as hypothesized previously [12]. Both LatB and Ory affected the lateral diffusion of PIN1-GFP in BY-2 cells [13], but since we have previously shown that lateral diffusion does not play a major role in the re-establishment of PIN2 polarity under normal conditions [10], we assume that it is not a major PIN polarizing factor upon LatB and/or Ory treatment, either. It has been theoretically predicted and experimentally verified that clathrin-mediated endocytosis plays a key role in PIN polarity regulation [9,10,[14][15][16][17][18]. An endocytosis-dependent, cytoskeleton-independent system based on differential rates of PIN endocytosis at different PM domains is therefore a prime candidate for the main mechanism of PIN polarity establishment. In the case of apically localized PIN2, such a mechanism might, in principle, work in two different ways, or their combination: A) Endocytosis machinery might be generally less active at the apical PM compared to the rest of the cell, or B) PIN2 molecules in the apical domain could be specifically "protected" from Biomolecules 2019, 9, 222 5 of 7 endocytosis by an unknown factor. The observation that the CLC-GFP signal is enhanced at the lateral PM domains has already indirectly hinted at mechanism A) [15], while the existence or nature of any factor specifically modulating PIN2 endocytosis in a domain-specific manner, as predicted in B), is so far elusive. It was proposed that NPH3-like proteins encoded by the MAB4/MEL gene family might possess this function, but the evidence presented to support this claim is far from conclusive [19].

Conclusions
In conclusion, we have demonstrated that apical PIN2 polarity can be generated and maintained in the absence of intact actin microfilaments and microtubules in both mitotic and interphase cells, as was previously also shown in the case of the basal polarity of PIN1 [8]. These observations exclude any cytoskeleton-guided vesicular trafficking-based mechanism as the main driving force of PIN polarity establishment. On the other hand, the substantial evidence that endocytosis plays a central role in PIN polarity [9,10,[14][15][16][17][18] necessitates that regulation of PIN endocytosis, and specifically its rates at different PM domains, be subjected to detailed analyses, as these will likely shed light on the still enigmatic mechanisms of cell polarity establishment in plants.