Applicability of non-invasive and live-cell holotomographic imaging on fungi.

The ability to acquire three-dimensional (3D) information of cellular structures without the need for fluorescent tags or staining makes holotomographic imaging a powerful tool in cellular biology. It provides valuable insights by measuring the refractive index (RI), an optical parameter describing the phase delay of light that passes through the living cell. Here, we demonstrate holotomographic imaging on industrial relevant ascomycete fungi and study their development and morphogenesis. This includes conidial germination, subcellular dynamics, and cytoplasmic flow during hyphal growth in Aspergillus niger. In addition, growth and budding of Aureobasidium pullulans cells are captured using holotomographic microscopy. Coupled to fluorescence imaging, lipid droplets, vacuoles, the mitochondrial network, and nuclei are targeted and analyzed in the 3D RI reconstructed images. While lipid droplets and vacuoles can be assigned to a specific RI pattern, mitochondria and nuclei were not pronounced. We show, that the lower sensitivity of RI measurements derives from the fungal cell wall that acts as an additional barrier for the illumination light of the microscope. After cell wall digest of hyphae and protoplast formation of A. niger expressing GFP-tagged histone H2A, location of nuclei could be determined by non-invasive RI measurements. Furthermore, we used coupled fluorescence microscopy to observe migration of nuclei in unperturbed hyphal segments and duplication during growth on a single-cell level. Detailed micromorphological studies in Saccharomyces cerevisiae and Trichoderma reesei are challenging due to cell size restrictions. Overall, holotomography opens up new avenues for exploring dynamic cellular processes in real time and enables the visualization of fungi from a new perspective.

Morphologically defined substages of tail morphogenesis in C. elegans males.

BACKGROUND: Sex-specific morphogenesis occurs in Caenorhabditis elegans in the vulva of the hermaphrodite and in the male tail during the last larval stage. Temporal progression of vulva morphogenesis has been described in fine detail. However, a similar precise description of male tail morphogenesis was lacking. RESULTS: We here describe morphogenesis of the male tail at time points matching vulva development with special focus on morphogenesis of the tail tip. Using fluorescent reporters, we follow changes in cell shapes, cell fusions, nuclear migration, modifications in the basement membrane, and formation of a new apical extracellular matrix at the end of the tail. CONCLUSION: Our analysis answers two open questions about tail tip morphogenesis (TTM) by showing that one of the four tail tip cells, hyp11, remains largely separate, while the other cells fully fuse with each other and with two additional tail cells to form a ventral tail syncytium. This merger of cells begins at the apical surface early during TTM but is only completed toward the end of the process. This work provides a framework for future investigations of cell biological factors that drive male tail morphogenesis.

FLN-2 functions in parallel to linker of nucleoskeleton and cytoskeleton complexes and CDC-42/actin pathways during P-cell nuclear migration through constricted spaces in Caenorhabditis elegans.

Nuclear migration through narrow constrictions is important for development, metastasis, and proinflammatory responses. Studies performed in tissue culture cells have implicated linker of nucleoskeleton and cytoskeleton (LINC) complexes, microtubule motors, the actin cytoskeleton, and nuclear envelope repair machinery as important mediators of nuclear movements through constricted spaces. However, little is understood about how these mechanisms operate to move nuclei in vivo. In Caenorhabditis elegans larvae, six pairs of hypodermal P cells migrate from lateral to ventral positions through a constricted space between the body wall muscles and the cuticle. P-cell nuclear migration is mediated in part by LINC complexes using a microtubule-based pathway and by an independent CDC-42/actin-based pathway. However, when both LINC complex and actin-based pathways are knocked out, many nuclei still migrate, suggesting the existence of additional pathways. Here, we show that FLN-2 functions in a third pathway to mediate P-cell nuclear migration. The predicted N-terminal actin-binding domain in FLN-2 that is found in canonical filamins is dispensable for FLN-2 function; this and structural predictions suggest that FLN-2 does not function as a filamin. The immunoglobulin-like repeats 4-8 of FLN-2 were necessary for P-cell nuclear migration. Furthermore, in the absence of the LINC complex component unc-84, fln-2 mutants had an increase in P-cell nuclear rupture. We conclude that FLN-2 functions to maintain the integrity of the nuclear envelope in parallel with the LINC complex and CDC-42/actin-based pathways to move P-cell nuclei through constricted spaces.

Reduction of cortical pulling at mitotic entry facilitates aster centration.

Equal cell division relies upon astral microtubule-based centering mechanisms, yet how the interplay between mitotic entry, cortical force generation and long astral microtubules leads to symmetric cell division is not resolved. We report that a cortically located sperm aster displaying long astral microtubules that penetrate the whole zygote does not undergo centration until mitotic entry. At mitotic entry, we find that microtubule-based cortical pulling is lost. Quantitative measurements of cortical pulling and cytoplasmic pulling together with physical simulations suggested that a wavelike loss of cortical pulling at mitotic entry leads to aster centration based on cytoplasmic pulling. Cortical actin is lost from the cortex at mitotic entry coincident with a fall in cortical tension from ∼300pN/µm to ∼100pN/µm. Following the loss of cortical force generators at mitotic entry, long microtubule-based cytoplasmic pulling is sufficient to displace the aster towards the cell center. These data reveal how mitotic aster centration is coordinated with mitotic entry in chordate zygotes.

ESCRT-I protein UBAP1 controls ventricular expansion and cortical neurogenesis via modulating adherens junctions of radial glial cells.

Intricate cerebral cortex formation is orchestrated by the precise behavior and division dynamics of radial glial cells (RGCs). Endocytosis functions in the recycling and remodeling of adherens junctions (AJs) in response to changes in RGC activity and function. Here, we show that conditional disruption of ubiquitin-associated protein 1 (UBAP1), a component of endosomal sorting complex required for transport (ESCRT), causes severe brain dysplasia and prenatal ventriculomegaly. UBAP1 depletion disrupts the AJs and polarity of RGCs, leading to failure of apically directed interkinetic nuclear migration. Accordingly, UBAP1 knockout or knockdown results in reduced proliferation and precocious differentiation of neural progenitor cells. Mechanistically, UBAP1 regulates the expression and surface localization of cell adhesion molecules, and ß-catenin over-expression significantly rescues the phenotypes of Ubap1 knockdown in vivo. Our study reveals a critical physiological role of the ESCRT machinery in cortical neurogenesis by regulating AJs of RGCs.

Percutaneous Endoscopic Transforaminal Discectomy for the Treatment of Lumbar Disc Herniation with Different Migration Levels: A Retrospective Study.

Objective: To investigate the surgical method and efficacy of percutaneous endoscopic transforaminal discectomy (PETD) for the treatment of lumbar disc herniation (LDH) with different migration levels by introducing the strategy of foramenoplasty with the "distal nucleus pulposus as the core". Methods: Clinical data of LDH patients who underwent single-segment PETD surgery were retrospectively analyzed. Three groups were categorized according to the degree of nucleus pulposus migration in the sagittal position: no migration group, mild migration group, and high migration group. Different sites of foramenoplasty were used for LDH with different degrees of migration. All patients were followed up for at least 12 months. The clinical and follow-up data of the three groups were compared. Results: A total of 102 patients were included, of which 46 (45.1%) were in the no migration group, 36 (35.3%) in the mild migration group, and 20 (19.6%) in the high migration group. Encouraging treatment results were obtained in all three groups. Conclusion: PETD is effective in the treatment of LDH with different degrees of migration, and the foramenoplasty concept of "distal nucleus pulposus as the core" can effectively guide the molding site of foramenoplasty and facilitate the accurate placement of the working trocar.

Novel lissencephaly-associated NDEL1 variant reveals distinct roles of NDE1 and NDEL1 in nucleokinesis and human cortical malformations.

The development of the cerebral cortex involves a series of dynamic events, including cell proliferation and migration, which rely on the motor protein dynein and its regulators NDE1 and NDEL1. While the loss of function in NDE1 leads to microcephaly-related malformations of cortical development (MCDs), NDEL1 variants have not been detected in MCD patients. Here, we identified two patients with pachygyria, with or without subcortical band heterotopia (SBH), carrying the same de novo somatic mosaic NDEL1 variant, p.Arg105Pro (p.R105P). Through single-cell RNA sequencing and spatial transcriptomic analysis, we observed complementary expression of Nde1/NDE1 and Ndel1/NDEL1 in neural progenitors and post-mitotic neurons, respectively. Ndel1 knockdown by in utero electroporation resulted in impaired neuronal migration, a phenotype that could not be rescued by p.R105P. Remarkably, p.R105P expression alone strongly disrupted neuronal migration, increased the length of the leading process, and impaired nucleus-centrosome coupling, suggesting a failure in nucleokinesis. Mechanistically, p.R105P disrupted NDEL1 binding to the dynein regulator LIS1. This study identifies the first lissencephaly-associated NDEL1 variant and sheds light on the distinct roles of NDE1 and NDEL1 in nucleokinesis and MCD pathogenesis.

Morphologically defined substages of tail morphogenesis in C. elegans males.

Background: Sex-specific morphogenesis occurs in C. elegans in the vulva of the hermaphrodite and in the male tail during the last larval stage. Temporal progression of vulva morphogenesis has been described in fine detail. However, a similar precise description of male tail morphogenesis was lacking. Results: We here describe morphogenesis of the male tail at time points matching vulva development with special focus on morphogenesis of the tail tip. Using fluorescent reporters, we follow changes in cell shapes, cell fusions, nuclear migration, modifications in the basement membrane and formation of a new apical extracellular matrix at the end of the tail. Conclusion: Our analysis answers two open questions about tail tip morphogenesis (TTM) by showing that one of the four tail tip cells, hyp11, remains separate while the other cells fuse with each other and with two additional tail cells to form a ventral tail syncytium. This fusion begins early during TTM but is only completed towards the end of the process. This work provides a framework for future investigations of cell-biological factors that drive male tail morphogenesis.

Actin and CDC-42 contribute to nuclear migration through constricted spaces in C. elegans.

Successful nuclear migration through constricted spaces between cells or in the extracellular matrix relies on the ability of the nucleus to deform. Little is known about how this takes place in vivo. We have studied confined nuclear migration in Caenorhabditis elegans larval P cells, which is mediated by the LINC complex to pull nuclei towards the minus ends of microtubules. Null mutations of the LINC component unc-84 lead to a temperature-dependent phenotype, suggesting a parallel pathway for P-cell nuclear migration. A forward genetic screen for enhancers of unc-84 identified cgef-1 (CDC-42 guanine nucleotide exchange factor). Knockdown of CDC-42 in the absence of the LINC complex led to a P-cell nuclear migration defect. Expression of constitutively active CDC-42 partially rescued nuclear migration in cgef-1; unc-84 double mutants, suggesting that CDC-42 functions downstream of CGEF-1. The Arp2/3 complex and non-muscle myosin II (NMY-2) were also found to function parallel to the LINC pathway. In our model, CGEF-1 activates CDC-42, which induces actin polymerization through the Arp2/3 complex to deform the nucleus during nuclear migration, and NMY-2 helps to push the nucleus through confined spaces.

Physcomitrium patens SUN2 Mediates MTOC Association with the Nuclear Envelope and Facilitates Chromosome Alignment during Spindle Assembly.

Plant cells lack centrosomes and instead utilize acentrosomal microtubule organizing centers (MTOCs) to rapidly increase the number of microtubules at the onset of spindle assembly. Although several proteins required for MTOC formation have been identified, how the MTOC is positioned at the right place is not known. Here, we show that the inner nuclear membrane protein SUN2 is required for MTOC association with the nuclear envelope (NE) during mitotic prophase in the moss Physcomitrium patens. In actively dividing protonemal cells, microtubules accumulate around the NE during prophase. In particular, regional MTOC is formed at the apical surface of the nucleus. However, microtubule accumulation around the NE was impaired and apical MTOCs were mislocalized in sun2 knockout cells. Upon NE breakdown, the mitotic spindle was assembled with mislocalized MTOCs. However, completion of chromosome alignment in the spindle was delayed; in severe cases, the chromosome was transiently detached from the spindle body. SUN2 tended to localize to the apical surface of the nucleus during prophase in a microtubule-dependent manner. Based on these results, we propose that SUN2 facilitates the attachment of microtubules to chromosomes during spindle assembly by localizing microtubules to the NE. MTOC mispositioning was also observed during the first division of the gametophore tissue. Thus, this study suggests that microtubule-nucleus linking, a well-known function of SUN in animals and yeast, is conserved in plants.

Live Cell Imaging of Dynamic Processes in Adult Zebrafish Retinal Cross-Section Cultures.

Following retinal injury, zebrafish possess the remarkable capacity to endogenously regenerate lost retinal neurons from Müller glia-derived neuronal progenitor cells. Additionally, neuronal cell types that are undamaged and persist in the injured retina are also produced. Thus, the zebrafish retina is an excellent system to study the integration of all neuronal cell types into an existing neuronal circuit. The few studies that examined axonal/dendritic outgrowth and the establishment of synaptic contacts by regenerated neurons predominantly utilized fixed tissue samples. We recently established a flatmount culture model to monitor Müller glia nuclear migration in real time by two-photon microscopy. However, in retinal flatmounts, z-stacks of the entire retinal z-dimension have to be acquired to image cells that extend through parts or the entirety of the neural retina, such as bipolar cells and Müller glia, respectively. Cellular processes with fast kinetics might thus be missed. Therefore, we generated a retinal cross-section culture from light-damaged zebrafish to image the entire Müller glia in one z-plane. Isolated dorsal retinal hemispheres were cut into two dorsal quarters and mounted with the cross-section view facing the coverslips of culture dishes, which allowed monitoring Müller glia nuclear migration using confocal microscopy. Confocal imaging of cross-section cultures is ultimately also applicable to live cell imaging of axon/dendrite formation of regenerated bipolar cells, while the flatmount culture model will be more suitable to monitor axon outgrowth of ganglion cells.

Using Budding Yeast as a Model to Understand Dynein-Mediated Cargo Transport.

Cytoplasmic dynein-1 is a minus end-directed microtubule motor that transports numerous cargoes in cell types throughout the evolutionary spectrum. Dynein is regulated by various motor-intrinsic and motor-extrinsic factors that enhance its processivity, recruit it to various cellular sites, or otherwise promote or restrict its activity. Studying dynein activity in higher eukaryotes is complicated by various factors, including the myriad functions in which this motor participates, and the consequential pleotropic effects associated with disrupting its activity. Budding yeast has long been a powerful model system for understanding this enormous motor protein complex, which is highly conserved between yeast and humans at the primary sequence and structural levels. Studies in budding yeast are simplified by the fact that dynein only performs one known function in this organism: to position the mitotic spindle at the site of cell division. Monitoring dynein-mediated spindle movements in budding yeast provides a powerful tool for the quantitative measurements of various motility parameters, and a system with which to assess the consequence of mutations in dynein or its regulators. Here, we provide detailed protocols to perform quantitative measurements of dynein activity in live cells using a combination of fluorescence microscopy and computational methods to track and quantitate dynein-mediated spindle movements. These methods are broadly applicable to anyone that wishes to perform fluorescence microscopy on budding yeast.

Assessment of Dynein-Mediated Nuclear Migration in the Developing Cortex by Live-Tissue Microscopy.

During development of the cerebral cortex, neuroepithelial and radial glial cells undergo an oscillatory nuclear movement throughout their cell cycle, termed interkinetic nuclear migration. The nucleus of postmitotic neurons derived from these neural stem cells also translocates in a saltatory manner to enable neuronal migration toward the cortical plate. In these processes, various molecular motors, including cytoplasmic dynein, myosin II, and kinesins, are the driving force for nuclear migration at different stages. Despite efforts made to understand the mechanism regulating cortical development over decades, novel gene mutations discovered in neurodevelopmental disorders indicate that missing pieces still remain. Gene manipulation by in utero electroporation combined with live microscopy of neural stem cells in brain slices provides a powerful method to capture their detailed behaviors during proliferation and migration. The procedures described in this chapter enable the monitoring of cell cycle progression, mitosis, morphological changes, and migratory patterns in situ. This approach facilitates the elucidation of gene functions in cortical development and neurodevelopmental disorders.

Synchronisation of apical constriction and cell cycle progression is a conserved behaviour of pseudostratified neuroepithelia informed by their tissue geometry.

Neuroepithelial cells balance tissue growth requirement with the morphogenetic imperative of closing the neural tube. They apically constrict to generate mechanical forces which elevate the neural folds, but are thought to apically dilate during mitosis. However, we previously reported that mitotic neuroepithelial cells in the mouse posterior neuropore have smaller apical surfaces than non-mitotic cells. Here, we document progressive apical enrichment of non-muscle myosin-II in mitotic, but not non-mitotic, neuroepithelial cells with smaller apical areas. Live-imaging of the chick posterior neuropore confirms apical constriction synchronised with mitosis, reaching maximal constriction by anaphase, before division and re-dilation. Mitotic apical constriction amplitude is significantly greater than interphase constrictions. To investigate conservation in humans, we characterised early stages of iPSC differentiation through dual SMAD-inhibition to robustly produce pseudostratified neuroepithelia with apically enriched actomyosin. These cultured neuroepithelial cells achieve an equivalent apical area to those in mouse embryos. iPSC-derived neuroepithelial cells have large apical areas in G2 which constrict in M phase and retain this constriction in G1/S. Given that this differentiation method produces anterior neural identities, we studied the anterior neuroepithelium of the elevating mouse mid-brain neural tube. Instead of constricting, mid-brain mitotic neuroepithelial cells have larger apical areas than interphase cells. Tissue geometry differs between the apically convex early midbrain and flat posterior neuropore. Culturing human neuroepithelia on equivalently convex surfaces prevents mitotic apical constriction. Thus, neuroepithelial cells undergo high-amplitude apical constriction synchronised with cell cycle progression but the timing of their constriction if influenced by tissue geometry.

Cephalopod retinal development shows vertebrate-like mechanisms of neurogenesis.

Coleoid cephalopods, including squid, cuttlefish, and octopus, have large and complex nervous systems and high-acuity, camera-type eyes. These traits are comparable only to features that are independently evolved in the vertebrate lineage. The size of animal nervous systems and the diversity of their constituent cell types is a result of the tight regulation of cellular proliferation and differentiation in development. Changes in the process of development during evolution that result in a diversity of neural cell types and variable nervous system size are not well understood. Here, we have pioneered live-imaging techniques and performed functional interrogation to show that the squid Doryteuthis pealeii utilizes mechanisms during retinal neurogenesis that are hallmarks of vertebrate processes. We find that retinal progenitor cells in the squid undergo nuclear migration until they exit the cell cycle. We identify retinal organization corresponding to progenitor, post-mitotic, and differentiated cells. Finally, we find that Notch signaling may regulate both retinal cell cycle and cell fate. Given the convergent evolution of elaborate visual systems in cephalopods and vertebrates, these results reveal common mechanisms that underlie the growth of highly proliferative neurogenic primordia. This work highlights mechanisms that may alter ontogenetic allometry and contribute to the evolution of complexity and growth in animal nervous systems.

Phosphatidylinositol transfer protein/planar cell polarity axis regulates neocortical morphogenesis by supporting interkinetic nuclear migration.

The neocortex expands explosively during embryonic development. The earliest populations of neural stem cells (NSCs) form a thin pseudostratified epithelium whose contour determines that of the adult neocortex. Neocortical complexity is accompanied by disproportional expansion of the NSC layer in its tangential dimension to increase tissue surface area. How such disproportional expansion is controlled remains unknown. We demonstrate that a phosphatidylinositol transfer protein (PITP)/non-canonical Wnt planar cell polarity (ncPCP) signaling axis promotes tangential expansion of developing neocortex. PITP signaling supports trafficking of specific ncPCP receptors from the NSC Golgi system to potentiate actomyosin activity important for cell-cycle-dependent interkinetic nuclear migration (IKNM). In turn, IKNM promotes lateral dispersion of newborn NSCs and tangential growth of the cerebral wall. These findings clarify functional roles for IKNM in NSC biology and identify tissue dysmorphogenesis resulting from impaired IKNM as a factor in autism risk, developmental brain disabilities, and neural tube birth defects.

Mechanical constraints to cell-cycle progression in a pseudostratified epithelium.

As organs and tissues approach their normal size during development or regeneration, growth slows down, and cell proliferation progressively comes to a halt. Among the various processes suggested to contribute to growth termination,1-10 mechanical feedback, perhaps via adherens junctions, has been suggested to play a role.11-14 However, since adherens junctions are only present in a narrow plane of the subapical region, other structures are likely needed to sense mechanical stresses along the apical-basal (A-B) axis, especially in a thick pseudostratified epithelium. This could be achieved by nuclei, which have been implicated in mechanotransduction in tissue culture.15 In addition, mechanical constraints imposed by nuclear crowding and spatial confinement could affect interkinetic nuclear migration (IKNM),16 which allows G2 nuclei to reach the apical surface, where they normally undergo mitosis.17-25 To explore how mechanical constraints affect IKNM, we devised an individual-based model that treats nuclei as deformable objects constrained by the cell cortex and the presence of other nuclei. The model predicts changes in the proportion of cell-cycle phases during growth, which we validate with the cell-cycle phase reporter FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator).26 However, this model does not preclude indefinite growth, leading us to postulate that nuclei must migrate basally to access a putative basal signal required for S phase entry. With this refinement, our updated model accounts for the observed progressive slowing down of growth and explains how pseudostratified epithelia reach a stereotypical thickness upon completion of growth.

Corrigendum: Serial Block-Face Scanning Electron Microscopy Reveals That Intercellular Nuclear Migration Occurs in Most Normal Tobacco Male Meiocytes.

[This corrects the article DOI: 10.3389/fpls.2021.672642.].

Intercellular nuclear migration in cryofixed tobacco male meiocytes.

In this study, intercellular nuclear migration (INM), also known as cytomixis, was documented in cryofixed plant meiocytes for the first time. Intact tobacco inflorescences and flower buds as well as dissected individual anthers were cryofixed in liquid nitrogen by plunge freezing. Cryosubstituted and cryosectioned male meiocytes were analyzed by light microscopy. For cryosubstitution, the frozen material was kept in acetic alcohol at - 70 °C for 1 week. For cryosectioning, the frozen material was sectioned at - 20 °C, and fixed with precooled acetic alcohol. Fixation of the intact tobacco inflorescences in Carnoy's solution was used as a control. Microscopy revealed good preservation of cell structure in the cryofixed anthers, flower buds, and inflorescences. INM was detectable in all the studied cryofixed and chemically fixed samples. The cytological picture of INM observed in the cryofixed meiocytes did not noticeably differ from the picture obtained with the chemically fixed cells. These results indicate that INM is observable irrespective of whether a physical or chemical fixation method is employed, with minimal damage from handling. Our results contradict the notion that INM is a phenomenon caused by mechanical, osmotic, or chemical artifacts during sample preparation.

Stalling interkinetic nuclear migration in curved pseudostratified epithelium of developing cochlea.

The bending of epithelial tubes is a fundamental process in organ morphogenesis, driven by various multicellular behaviours. The cochlea in the mammalian inner ear is a representative example of spiral tissue architecture where the continuous bending of the duct is a fundamental component of its morphogenetic process. Although the cochlear duct morphogenesis has been studied by genetic approaches extensively, it is still unclear how the cochlear duct morphology is physically formed. Here, we report that nuclear behaviour changes are associated with the curvature of the pseudostratified epithelium during murine cochlear development. Two-photon live-cell imaging reveals that the nuclei shuttle between the luminal and basal edges of the cell is in phase with cell-cycle progression, known as interkinetic nuclear migration, in the flat region of the pseudostratified epithelium. However, the nuclei become stationary on the luminal side following mitosis in the curved region. Mathematical modelling together with perturbation experiments shows that this nuclear stalling facilitates luminal-basal differential growth within the epithelium, suggesting that the nuclear stalling would contribute to the bending of the pseudostratified epithelium during the cochlear duct development. The findings suggest a possible scenario of differential growth which sculpts the tissue shape, driven by collective nuclear dynamics.