Cortical Microtubules Research Articles

Tissue distribution and subcellular localization of the family of Kidney Ankyrin Repeat Domain (KANK) proteins

Kidney Ankyrin Repeat-containing Proteins (KANKs) comprise a family of four evolutionary conserved proteins (KANK1 to 4) that localize to the belt of mature focal adhesions (FAs) where they regulate integrin-mediated adhesion, actomyosin contractility, and link FAs to the cortical microtubule stabilization complex (CMSC). The human KANK proteins were first identified in kidney and have been associated with kidney cancer and nephrotic syndrome. Here, we report the distributions and subcellular localizations of the four Kank mRNAs and proteins in mouse tissues. We found that the KANK family members display distinct and rarely overlapping expression patterns. Whereas KANK1 is expressed at the basal side of epithelial cells of all tissues tested, KANK2 expression is mainly observed at the plasma membrane and/or cytoplasm of mesenchymal cells and KANK3 exclusively in vascular and lymphatic endothelial cells. KANK4 shows the least widespread expression pattern and when present, overlaps with KANK2 in contractile cells, such as smooth muscle cells and pericytes. Our findings show that KANKs are widely expressed in a cell type-specific manner, which suggests that they have cell- and tissue-specific functions.

Ultrastructure and Cytochemistry of the Mature Spermatozoon of Khawia Armeniaca (Cholodkovsky, 1915) (Caryophyllidea: Lytocestidae), a Parasite of Capoeta Capoeta Sevangi (De Filippi, 1865) (Teleostei, Cyprinidae).

SummaryThe mature spermatozoon of Khawia armeniaca, a monozoic caryophyllidean parasite of templar fish Capoeta capoeta sevangi (De Filippi, 1865) from the Lake Sevan, Armenia, has been studied using transmission electron microscopy and cytochemical technique of Thiéry (1967) for the first time. The mature spermatozoon of K. armeniaca consists of a single axoneme with the 9+‘1’ trepaxonematan structure, cortical microtubules and nucleus which are situated parallel to the longitudinal axis of the spermatozoon, and a moderately electrondense cytoplasm with glycogen particles. The cortical microtubules are arranged in one continuous semicircle beneath the plasma membrane in Region II and anterior part of Region III of the mature spermatozoon. The two opposite rows of cortical microtubules are observed in the remaining nuclear and at the beginning of the postnuclear part (Regions III, IV) of the male gamete The number of cortical microtubules is remarkably variable in the spermatozoa of various Khawia species. K. armeniaca exhibits the highest number of cortical microtubules in comparison with K. sinensis and K. rossittensis. Glycogen was detected in the cytoplasm of prenuclear (II), nuclear (III) and postnuclear (IV) regions with different ultrastructural organization of the mature spermatozoon of K. armeniaca. Variations of sperm ultrastructural characters within caryophyllideans and other cestodes are discussed.

Effects of the protein GCP4 on gametophyte development in Arabidopsis thaliana.

γ-Tubulin complex protein 4 (GCP4, encoded by AT3G53760) participates in microtubule (MT) nucleation in Arabidopsis thaliana, affecting the MT nucleation angles in cortical MTs, and the formation of the spindle and phragmoplasts during mitosis. Here, we report that GCP4 plays a critical role in gametophyte development. The results indicate that the gcp4 mutant caused by T-DNA insertion may express an aberrant gene product interfering with normal GCP4 expression, ultimately leading to the formation of desiccated ovules and aborted seeds. An analysis of transmission efficiency (TE) indicated that female gametophytes were more impaired in development than male gametophytes, and so observation and analysis of gametophyte defects were conducted. Complementation lines obtained by the native promoter and GCP4-coded CDS gene sequence fused with GFP reduced the numbers of lethal phenotypes of the gcp4 mutant. The localization of GCP4 in the gametophyte was detected in cytoplasm around nuclei and in vicinity of plasma membrane of pollen grains, and also detected in full cytoplasm and around the nuclei of ovules in complementation line. Thus, it was established that GCP4 influences the functionality of gametophytes during gametophyte development.

ABNORMAL SHOOT 6 interacts with KATANIN 1 and SHADE AVOIDANCE 4 to promote cortical microtubule severing and ordering in Arabidopsis.

Plant interphase cortical microtubules (cMTs) mediate anisotropic cell expansion in response to environmental and developmental cues. In Arabidopsis thaliana, KATANIN 1 (KTN1), the p60 catalytic subunit of the conserved MT-severing enzyme katanin, is essential for cMT ordering and anisotropic cell expansion. However, the regulation of KTN1-mediated cMT severing and ordering remains unclear. In this work, we report that the Arabidopsis IQ67 DOMAIN (IQD) family gene ABNORMAL SHOOT 6 (ABS6) encodes a MT-associated protein. Overexpression of ABS6 leads to elongated cotyledons, directional pavement cell expansion, and highly ordered transverse cMT arrays. Genetic suppressor analysis revealed that ABS6-mediated cMT ordering is dependent on KTN1 and SHADE AVOIDANCE 4 (SAV4). Live imaging of cMT dynamics showed that both ABS6 and SAV4 function as positive regulators of cMT severing. Furthermore, ABS6 directly interacts with KTN1 and SAV4 and promotes their recruitment to the cMTs. Finally, analysis of loss-of-function mutant combinations showed that ABS6, SAV4, and KTN1 work together to ensure the robust ethylene response in the apical hook of dark-grown seedlings. Together, our findings establish ABS6 and SAV4 as positive regulators of cMT severing and ordering, and highlight the role of cMT dynamics in fine-tuning differential growth in plants.

Salt Stress-Induced Structural Changes Are Mitigated in Transgenic Tomato Plants Over-Expressing Superoxide Dismutase.

Simple SummaryWe analyzed the morphological changes in root tip cells caused by the application of iso-osmotic NaCl and Na2SO4 solutions to tomato plants harboring an introduced superoxide dismutase gene. To study the roots of tomato plants cultivar Belyi Naliv and FeSOD-transgenic line, we examined the distribution of reactive oxygen species and immunodetection of α-tubulin. The differences in the microtubules cortical network between wild type and transgenic plants without salinity were detected. The differences were found in the cortical network of microtubules between control and transgenic plants in the absence of salt stress. While an ordered microtubule network was revealed in the root cells of wild type tomato, no such degree of ordering was detected in transgenic line cells. The signs of microtubule disorganization in root cells of wild type plants were manifested under the NaCl and Na2SO4 treatment. On the contrary, the cytoskeleton structural organization in the transgenic line cells was more ordered. In addition, the formation of atypical tubulin polymers was observed in response to salt stress. Changes in cell size, due to both vacuolization and impaired cell expansion in columella zone and cap initials, were responsible for the root tip tissue modification.Various abiotic stresses cause the appearance of reactive oxygen species (ROS) in plant cells, which seriously damage the cellular structures. The engineering of transgenic plants with higher production of ROS-scavenging enzyme in plant cells could protect the integrity of such a fine intracellular structure as the cytoskeleton and each cellular compartment. We analyzed the morphological changes in root tip cells caused by the application of iso-osmotic NaCl and Na2SO4 solutions to tomato plants harboring an introduced superoxide dismutase gene. To study the roots of tomato plants cultivar Belyi Naliv (WT) and FeSOD-transgenic line, we examined the distribution of ROS and enzyme-linked immunosorbent detection of α-tubulin. In addition, longitudinal sections of the root apexes were compared. Transmission electronic microscopy of atypical cytoskeleton structures was also performed. The differences in the microtubules cortical network between WT and transgenic plants without salt stress were detected. The differences were found in the cortical network of microtubules between WT and transgenic plants in the absence of salt stress. While an ordered microtubule network was revealed in the root cells of WT tomato, no such degree of ordering was detected in transgenic line cells. The signs of microtubule disorganization in root cells of WT plants were manifested under the NaCl treatment. On the contrary, the cytoskeleton structural organization in the transgenic line cells was more ordered. Similar changes, including the cortical microtubules disorganization, possibly associated with the formation of atypical tubulin polymers as a response to salt stress caused by Na2SO4 treatment, were also observed. Changes in cell size, due to both vacuolization and impaired cell expansion in columella zone and cap initials, were responsible for the root tip tissue modification.

Rapid, redox-mediated mechanical susceptibility of the cortical microtubule lattice in skeletal muscle

The highly ordered cortical microtubule lattice of skeletal muscle is disorganized in dystrophin-deficient mdx mice. Implicated mechanisms include loss of dystrophin binding, altered α-tubulin posttranslational modification, expression of a β-tubulin involved in regeneration, and reactive oxygen species (ROS). Here we show that the transverse microtubules in mdx muscle expressing miniaturized dystrophins are rapidly lost after eccentric contraction. Analysis of mdx lines expressing different dystrophin constructs demonstrate that spectrin-like repeats R4-15 and R20-23 were required for mechanically stable microtubules. Microtubule loss was prevented by the non-specific antioxidant N-acetylcysteine while inhibition of NADPH oxidase 2 had only a partial effect, suggesting that ROS from multiple sources mediate the rapid loss of transverse microtubules after eccentric contraction. Finally, ablation of α-dystrobrevin, β- or γ-cytoplasmic actin phenocopied the transverse microtubule instability of miniaturized dystrophins. Our data demonstrate that multiple dystrophin domains, α-dystrobrevin and cytoplasmic actins are necessary for mechanically stable microtubules.

Microtubule-Mediated Wall Anisotropy Contributes to Leaf Blade Flattening

SummaryPlant organs can adopt a wide range of shapes, resulting from highly directional cell growth and divisions. We focus here on leaves and leaf-like organs in Arabidopsis and tomato, characterized by the formation of thin, flat laminae. Combining experimental approaches with 3D mechanical modeling, we provide evidence that leaf shape depends on cortical microtubule mediated cellulose deposition along the main predicted stress orientations, in particular, along the adaxial-abaxial axis in internal cell walls. This behavior can be explained by a mechanical feedback and has the potential to sustain and even amplify a preexisting degree of flatness, which in turn depends on genes involved in the control of organ polarity and leaf margin formation.

Mutations in the Pectin Methyltransferase QUASIMODO2 Influence Cellulose Biosynthesis and Wall Integrity in Arabidopsis.

Pectins are abundant in the cell walls of dicotyledonous plants, but how they interact with other wall polymers and influence wall integrity and cell growth has remained mysterious. Here, we verified that QUASIMODO2 (QUA2) is a pectin methyltransferase and determined that QUA2 is required for normal pectin biosynthesis. To gain further insight into how pectin affects wall assembly and integrity maintenance, we investigated cellulose biosynthesis, cellulose organization, cortical microtubules, and wall integrity signaling in two mutant alleles of Arabidopsis (Arabidopsis thaliana) QUA2, qua2 and tsd2 In both mutants, crystalline cellulose content is reduced, cellulose synthase particles move more slowly, and cellulose organization is aberrant. NMR analysis shows higher mobility of cellulose and matrix polysaccharides in the mutants. Microtubules in mutant hypocotyls have aberrant organization and depolymerize more readily upon treatment with oryzalin or external force. The expression of genes related to wall integrity, wall biosynthesis, and microtubule stability is dysregulated in both mutants. These data provide insights into how homogalacturonan is methylesterified upon its synthesis, the mechanisms by which pectin functionally interacts with cellulose, and how these interactions are translated into intracellular regulation to maintain the structural integrity of the cell wall during plant growth and development.

KATANIN-dependent mechanical properties of the stigmatic cell wall mediate the pollen tube path in Arabidopsis

Successful fertilization in angiosperms depends on the proper trajectory of pollen tubes through the pistil tissues to reach the ovules. Pollen tubes first grow within the cell wall of the papilla cells, applying pressure to the cell. Mechanical forces are known to play a major role in plant cell shape by controlling the orientation of cortical microtubules (CMTs), which in turn mediate deposition of cellulose microfibrils (CMFs). Here, by combining imaging, genetic and chemical approaches, we show that isotropic reorientation of CMTs and CMFs in aged Col-0 and katanin1-5 (ktn1-5) papilla cells is accompanied by a tendency of pollen tubes to coil around the papillae. We show that this coiled phenotype is associated with specific mechanical properties of the cell walls that provide less resistance to pollen tube growth. Our results reveal an unexpected role for KTN1 in pollen tube guidance on the stigma by ensuring mechanical anisotropy of the papilla cell wall.

Growing straight through walls.

The pollen tube in a flowering plant grows in a direction that is influenced by the mechanical properties of the stigma papillae and the organization of structures called cortical microtubules inside these cells.

Live-cell RESOLFT nanoscopy of transgenic Arabidopsis thaliana.

Subdiffraction super‐resolution fluorescence microscopy, or nanoscopy, has seen remarkable developments in the last two decades. Yet, for the visualization of plant cells, nanoscopy is still rarely used. In this study, we established RESOLFT nanoscopy on living green plant tissue. Live‐cell RESOLFT nanoscopy requires and utilizes comparatively low light doses and intensities to overcome the diffraction barrier. We generated a transgenic Arabidopsis thaliana plant line expressing the reversibly switchable fluorescent protein rsEGFP2 fused to the mammalian microtubule‐associated protein 4 (MAP4) in order to ubiquitously label the microtubule cytoskeleton. We demonstrate the use of RESOLFT nanoscopy for extended time‐lapse imaging of cortical microtubules in Arabidopsis leaf discs. By combining our approach with fluorescence lifetime gating, we were able to acquire live‐cell RESOLFT images even close to chloroplasts, which exhibit very strong autofluorescence. The data demonstrate the feasibility of subdiffraction resolution imaging in transgenic plant material with minimal requirements for sample preparation.

Auxin-induced signaling protein nanoclustering contributes to cell polarity formation

Cell polarity is fundamental to the development of both eukaryotes and prokaryotes, yet the mechanisms behind its formation are not well understood. Here we found that, phytohormone auxin-induced, sterol-dependent nanoclustering of cell surface transmembrane receptor kinase 1 (TMK1) is critical for the formation of polarized domains at the plasma membrane (PM) during the morphogenesis of cotyledon pavement cells (PC) in Arabidopsis. Auxin-induced TMK1 nanoclustering stabilizes flotillin1-associated ordered nanodomains, which in turn promote the nanoclustering of ROP6 GTPase that acts downstream of TMK1 to regulate cortical microtubule organization. In turn, cortical microtubules further stabilize TMK1- and flotillin1-containing nanoclusters at the PM. Hence, we propose a new paradigm for polarity formation: A diffusive signal triggers cell polarization by promoting cell surface receptor-mediated nanoclustering of signaling components and cytoskeleton-mediated positive feedback that reinforces these nanodomains into polarized domains.

Spermatological characters in the Lepocreadioidea, with first data on Holorchis pycnoporus (Aephnidiogenidae), a parasite of the Striped seabream Lithognathus mormyrus (Sparidae) from the Gulf of Gabes (Tunisia)

The ultrastructural characteristics of the mature spermatozoon of Holorchis pycnoporus (Digenea, Lepocreadioidea, Aephnidiogenidae) are described by means of transmission electron microscopy (TEM). Live worms were collected from the digestive tract of the Striped seabream Lithognathus mormyrus (Teleostei, Sparidae), off the Gulf of Gabès at La Chebba (Tunisia). The ultrastructural study reveals that the male gamete of H. pycnoporus is a filiform cell tapered at both extremities and exhibiting the type III of the digenean spermatozoon proposed by Bakhoum et al. (2017a), characterized by the presence of (1) two axonemes with the 9 + ‘1’ pattern of the Trepaxonemata, (2) external ornamentation of the plasma membrane located in a posterior part of the anterior region of the spermatozoon and associated with cortical microtubules, (3) two bundles of parallel cortical microtubules with maximum number located in the middle part of the spermatozoon, and (4) generally two mitochondria. Moreover, H. pycnoporus shares a set of ultrastructural characteristics with the studied Aephnidiogenidae such as: (1) two 9+'1' axonemes of different lengths, (2) an anterior electron-dense material, (3) mitochondrion/a, (4) an external ornamentation of the plasma membrane associated with cortical microtubules, and (5) two bundles of parallel cortical microtubules with their maximum number (around 24 microtubules) located in the middle or posterior part of the spermatozoon. In the Aephnidiogenidae, the mature spermatozoon exhibits a similar ultrastructural pattern. Some differences are observed, particularly the location of maximum number of cortical microtubules and the number of mitochondria. The presence of the anterolateral electron-dense material is the major particularity in species belonging to the Lepocreadioidea. This anterior dense material could be a synapomorphy for the superfamily and an ultrastructural argument supporting the monophyletic status of the Lepocreadioidea (Bray and Cribb, 2012).

CELLULOSE SYNTHASE INTERACTING 1 is required for wood mechanics and leaf morphology in aspen.

Cellulose microfibrils synthesized by CELLULOSE SYNTHASE COMPLEXES (CSCs) are the main load-bearing polymers in wood. CELLULOSE SYNTHASE INTERACTING1 (CSI1) connects CSCs with cortical microtubules, which align with cellulose microfibrils. Mechanical properties of wood are dependent on cellulose microfibril alignment and structure in the cell walls, but the molecular mechanism(s) defining these features is unknown. Herein, we investigated the role of CSI1 in hybrid aspen (Populus tremula×Populus tremuloides) by characterizing transgenic lines with significantly reduced CSI1 transcript abundance. Reduction in leaves (50-80%) caused leaf twisting and misshaped pavement cells, while reduction (70-90%) in developing xylem led to impaired mechanical wood properties evident as a decrease in the elastic modulus and rupture. X-ray diffraction measurements indicate that microfibril angle was not impacted by the altered CSI1 abundance in developing wood fibres. Instead, the augmented wood phenotype of the transgenic trees was associated with a reduced cellulose degree of polymerization. These findings establish a function for CSI1 in wood mechanics and in defining leaf cell shape. Furthermore, the results imply that the microfibril angle in wood is defined by CSI1 independent mechanism(s).

Giant ankyrin-B suppresses stochastic collateral axon branching through direct interaction with microtubules.

Giant ankyrin-B (gAnkB) is a 440-kD neurospecific ankyrin-B isoform and a high-confidence target for autism mutations. gAnkB suppresses axon branching through coordination of cortical microtubules, and autism-related mutation of gAnkB results in ectopic neuronal connectivity. We identified a bipartite motif from gAnkB, which bundles and avidly binds to microtubules in vitro. This motif is composed of a module of 15 tandem repeats followed by a short, conserved fragment also found in giant ankyrin-G (BG-box). Combination of these two parts synergistically increases microtubule-binding avidity. Transfection of astrocytes (which lack gAnkB) with WT gAnkB resulted in prominent bundling of microtubules, which did not occur with mutant gAnkB with impaired microtubule-binding activity. Similarly, rescue of gAnkB-deficient neurons with WT gAnkB suppressed axonal branching and invasion of EB3-tagged microtubules into filopodia, which did not occur with the same mutant gAnkB. Together, these findings demonstrate that gAnkB suppresses axon collateral branching and prevents microtubule invasion of nascent axon branches through direct interaction with microtubules.

Associations between phytohormones and cellulose biosynthesis in land plants.

Phytohormones are small molecules that regulate virtually every aspect of plant growth and development, from basic cellular processes, such as cell expansion and division, to whole plant environmental responses. While the phytohormone levels and distribution thus tell the plant how to adjust itself, the corresponding growth alterations are actuated by cell wall modification/synthesis and internal turgor. Plant cell walls are complex polysaccharide-rich extracellular matrixes that surround all plant cells. Among the cell wall components, cellulose is typically the major polysaccharide, and is the load-bearing structure of the walls. Hence, the cell wall distribution of cellulose, which is synthesized by large Cellulose Synthase protein complexes at the cell surface, directs plant growth. Here, we review the relationships between key phytohormone classes and cellulose deposition in plant systems. We present the core signalling pathways associated with each phytohormone and discuss the current understanding of how these signalling pathways impact cellulose biosynthesis with a particular focus on transcriptional and post-translational regulation. Because cortical microtubules underlying the plasma membrane significantly impact the trajectories of Cellulose Synthase Complexes, we also discuss the current understanding of how phytohormone signalling impacts the cortical microtubule array. Given the importance of cellulose deposition and phytohormone signalling in plant growth and development, one would expect that there is substantial cross-talk between these processes; however, mechanisms for many of these relationships remain unclear and should be considered as the target of future studies.

Articles of Significant Interest in This Issue

Human immunodeficiency virus type 1 (HIV-1) exploits specialized microtubule (MT) plus-end tracking proteins (commonly known as +TIPs) to induce the formation of stable MTs soon after virus entry and to promote early stages of infection. Mitra et al. (e00404-20) found that distinct domains in cytoplasmic linker-associated protein 2 (CLASP2), a +TIP that captures cortical MT plus ends to enable filament stabilization, mediate interactions with incoming HIV-1 particles, and facilitate MT stabilization and trafficking of incoming cores. These findings identify a new +TIP that acts as a host cofactor to promote early stages of viral infection.

Ultrastructure of the spermatozoon of Lecithostaphylus parexocoeti (Digenea, Microphalloidea, Zoogonidae) parasite of the flying fish Cheilopogon pinnatibarbatus (Teleostei, Exocoetidae) off Senegal and their implication on the phylogenetic relationships in Microphalloidea

This study shows the ultrastructure of the mature spermatozoon of Lecithostaphylus parexocoeti (Digenea, Microphalloidea, Zoogonidae) parasite of the flying fish Cheilopogon pinnatibarbatus (Exocoetidae) captured in the Atlantic Ocean near Dakar (Senegal). This is the first report of L. parexocoeti from Senegal and from this host species. The mature spermatozoon is filiform and exhibits two axonemes of the 9+“1” pattern of the Trepaxonemata, an external ornamentation associated with spine-like bodies, parallel cortical microtubules, a nucleus, two mitochondria and granules of glycogen. The particularities of the spermatozoon of L. parexocoeti are the location of the first mitochondrion and the morphology of its anterior extremity with only cortical microtubules and external ornamentations of the plasma membrane. To our knowledge, this type of anterior extremity has never been described in the superfamily Microphalloidea. However, the ultrastructure of the posterior extremity of the spermatozoon corresponds to the fasciolidean type that was previously described in the Zoogonidae.

Spatiotemporal Pattern of Ectopic Cell Divisions Contribute to Mis-Shaped Phenotype of Primary and Lateral Roots of katanin1 Mutant.

Pattern formation, cell proliferation, and directional cell growth, are driving factors of plant organ shape, size, and overall vegetative development. The establishment of vegetative morphogenesis strongly depends on spatiotemporal control and synchronization of formative and proliferative cell division patterns. In this context, the progression of cell division and the regulation of cell division plane orientation are defined by molecular mechanisms converging to the proper positioning and temporal reorganization of microtubule arrays such as the preprophase microtubule band, the mitotic spindle and the cytokinetic phragmoplast. By focusing on the tractable example of primary root development and lateral root emergence in Arabidopsis thaliana, genetic studies have highlighted the importance of mechanisms underlying microtubule reorganization in the establishment of the root system. In this regard, severe alterations of root growth, and development found in extensively studied katanin1 mutants of A. thaliana (fra2, lue1, and ktn1-2), were previously attributed to defective rearrangements of cortical microtubules and aberrant cell division plane reorientation. How KATANIN1-mediated microtubule severing contributes to tissue patterning and organ morphogenesis, ultimately leading to anisotropy in microtubule organization is a trending topic under vigorous investigation. Here we addressed this issue during root development, using advanced light-sheet fluorescence microscopy (LSFM) and long-term imaging of ktn1-2 mutant expressing the GFP-TUA6 microtubule marker. This method allowed spatial and temporal monitoring of cell division patterns in growing roots. Analysis of acquired multidimensional data sets revealed the occurrence of ectopic cell divisions in various tissues including the calyptrogen and the protoxylem of the main root, as well as in lateral root primordia. Notably the ktn1-2 mutant exhibited excessive longitudinal cell divisions (parallel to the root axis) at ectopic positions. This suggested that changes in the cell division pattern and the occurrence of ectopic cell divisions contributed significantly to pleiotropic root phenotypes of ktn1-2 mutant. LSFM provided evidence that KATANIN1 is required for the spatiotemporal control of cell divisions and establishment of tissue patterns in living A. thaliana roots.

Complementary Superresolution Visualization of Composite Plant Microtubule Organization and Dynamics.

Microtubule bundling is an essential mechanism underlying the biased organization of interphase and mitotic microtubular systems of eukaryotes in ordered arrays. Microtubule bundle formation can be exemplified in plants, where the formation of parallel microtubule systems in the cell cortex or the spindle midzone is largely owing to the microtubule crosslinking activity of a family of microtubule associated proteins, designated as MAP65s. Among the nine members of this family in Arabidopsis thaliana, MAP65-1 and MAP65-2 are ubiquitous and functionally redundant. Crosslinked microtubules can form high-order arrays, which are difficult to track using widefield or confocal laser scanning microscopy approaches. Here, we followed spatiotemporal patterns of MAP65-2 localization in hypocotyl cells of Arabidopsis stably expressing fluorescent protein fusions of MAP65-2 and tubulin. To circumvent imaging difficulties arising from the density of cortical microtubule bundles, we use different superresolution approaches including Airyscan confocal laser scanning microscopy (ACLSM), structured illumination microscopy (SIM), total internal reflection SIM (TIRF-SIM), and photoactivation localization microscopy (PALM). We provide insights into spatiotemporal relations between microtubules and MAP65-2 crossbridges by combining SIM and ACLSM. We obtain further details on MAP65-2 distribution by single molecule localization microscopy (SMLM) imaging of either mEos3.2-MAP65-2 stochastic photoconversion, or eGFP-MAP65-2 stochastic emission fluctuations under specific illumination conditions. Time-dependent dynamics of MAP65-2 were tracked at variable time resolution using SIM, TIRF-SIM, and ACLSM and post-acquisition kymograph analysis. ACLSM imaging further allowed to track end-wise dynamics of microtubules labeled with TUA6-GFP and to correlate them with concomitant fluctuations of MAP65-2 tagged with tagRFP. All different microscopy modules examined herein are accompanied by restrictions in either the spatial resolution achieved, or in the frame rates of image acquisition. PALM imaging is compromised by speed of acquisition. This limitation was partially compensated by exploiting emission fluctuations of eGFP which allowed much higher photon counts at substantially smaller time series compared to mEos3.2. SIM, TIRF-SIM, and ACLSM were the methods of choice to follow the dynamics of MAP65-2 in bundles of different complexity. Conclusively, the combination of different superresolution methods allowed for inferences on the distribution and dynamics of MAP65-2 within microtubule bundles of living A. thaliana cells.