Collapse of late endosomal pH elicits a rapid Rab7 response via the V-ATPase and RILP.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of the correct pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). Here, we treated mammalian cells with the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyperactivation of Rab7 (herein referring to Rab7a), and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR; also known as IGF2R) recycling on pH-neutralized LEs. pH neutralization (NH4Cl) and expression of Rab7 hyperactive mutants alone can both phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit (encoded by ATP6V1G1) of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds and in disease states.

Chromosomal passenger complex condensates generate parallel microtubule bundles in vitro.

The mitotic spindle contains many bundles of microtubules (MTs) including midzones and kinetochore fibers, but little is known about how bundled structures are formed. Here, we show that the chromosomal passenger complex (CPC) purified from Escherichia coli undergoes liquid-liquid demixing in vitro. An emergent property of the resultant condensates is to generate parallel MT bundles when incubated with free tubulin and GTP in vitro. We demonstrate that MT bundles emerge from CPC droplets with protruding minus ends that then grow into long and tapered MT structures. During this growth, we found that the CPC in these condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for liquid-liquid demixing or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data demonstrate that an in vitro biochemical activity to produce MT bundles emerges after the concentration of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle. Moreover, these data suggest that cells contain MT-organizing centers that generate MT bundles that emerge with the opposite polarity from centrosomes.

Mechanics of the spindle apparatus.

During mitosis microtubules self-organize to form a bipolar mitotic spindle structure, which positions the sister chromatids on the spindle mid-plane and separates them afterwards. Previous studies have identified many spindle associated proteins. Yet, we do not fully understand how these nanoscopic proteins lead to force generation through interactions of individual microtubules, motor proteins and chromosomes, and how a large number of these local interactions ultimately determine the structure and mechanics of the spindle in micron scale. Here we review the current understanding and open questions related to the structure and mechanics of the mitotic spindle. We then discuss how a combination of electron microscopy and computational modeling can be used to tackle some of these open questions.

Semi-automatic stitching of filamentous structures in image stacks from serial-section electron tomography.

We present a software-assisted workflow for the alignment and matching of filamentous structures across a three-dimensional (3D) stack of serial images. This is achieved by combining automatic methods, visual validation, and interactive correction. After the computation of an initial automatic matching, the user can continuously improve the result by interactively correcting landmarks or matches of filaments. Supported by a visual quality assessment of regions that have been already inspected, this allows a trade-off between quality and manual labour. The software tool was developed in an interdisciplinary collaboration between computer scientists and cell biologists to investigate cell division by quantitative 3D analysis of microtubules (MTs) in both mitotic and meiotic spindles. For this, each spindle is cut into a series of semi-thick physical sections, of which electron tomograms are acquired. The serial tomograms are then stitched and non-rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. In practice, automatic stitching alone provides only an incomplete solution, because large physical distortions and a low signal-to-noise ratio often cause experimental difficulties. To derive 3D models of spindles despite dealing with imperfect data related to sample preparation and subsequent data collection, semi-automatic validation and correction is required to remove stitching mistakes. However, due to the large number of MTs in spindles (up to 30k) and their resulting dense spatial arrangement, a naive inspection of each MT is too time-consuming. Furthermore, an interactive visualisation of the full image stack is hampered by the size of the data (up to 100 GB). Here, we present a specialised, interactive, semi-automatic solution that considers all requirements for large-scale stitching of filamentous structures in serial-section image stacks. To the best of our knowledge, it is the only currently available tool which is able to process data of the type and size presented here. The key to our solution is a careful design of the visualisation and interaction tools for each processing step to guarantee real-time response, and an optimised workflow that efficiently guides the user through datasets. The final solution presented here is the result of an iterative process with tight feedback loops between the involved computer scientists and cell biologists. LAY DESCRIPTION: Electron tomography of biological samples is used for a three-dimensional (3D) reconstruction of filamentous structures, such as microtubules (MTs) in mitotic and meiotic spindles. Large-scale electron tomography can be applied to increase the reconstructed volume for the visualisation of full spindles. For this, each spindle is cut into a series of semi-thick physical sections, from which electron tomograms are acquired. The serial tomograms are then stitched and non-rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. Previously, we presented fully automatic approaches for this 3D reconstruction pipeline. However, large volumes often suffer from imperfections (ie physical distortions) caused by the image acquisition process, making it difficult to apply fully automatic approaches for matching and stitching of numerous tomograms. Therefore, we developed an interactive, semi-automatic solution that considers all requirements for large-scale stitching of microtubules in image stacks of consecutive sections. We achieved this by combining automatic methods, visual validation and interactive error correction, thus allowing the user to continuously improve the result by interactively correcting landmarks or matches of filaments. We present large-scale reconstructions of spindles in which the automatic workflow failed and where different steps of manual corrections were needed. Our approach is also applicable to other biological samples showing 3D distributions of MTs in a number of different cellular contexts.

Mitotic spindles revisited - new insights from 3D electron microscopy.

The mitotic spindle is a complex three-dimensional (3D) apparatus that functions to ensure the faithful segregation of chromosomes during cell division. Our current understanding of spindle architecture is mainly based on a plethora of information derived from light microscopy with rather few insights about spindle ultrastructure obtained from electron microscopy. In this Review, we will provide insights into the history of imaging of mitotic spindles and highlight recent technological advances in electron tomography and data processing, which have delivered detailed 3D reconstructions of mitotic spindles in the early embryo of the nematode Caenorhabditis elegans Tomographic reconstructions provide novel views on spindles and will enable us to revisit and address long-standing questions in the field of mitosis.

Positioning of Particles in Active Droplets.

Chemically active droplets are nonequilibrium systems that combine phase separation with chemical reactions. We here investigate how the activity introduced by the chemical reactions influences solid particles inside such droplets. We find that passive particles are centered in active droplets governed by first-order reactions. In autocatalytic active droplets, only catalytically active particles can be centered. An example of such systems in biology are centrosomes. Our study can account for the observed positioning of centrioles and provides a general mechanism to control the position of particles within chemically active droplets.

The Mitotic Spindle in the One-Cell C. elegans Embryo Is Positioned with High Precision and Stability.

Precise positioning of the mitotic spindle is important for specifying the plane of cell division, which in turn determines how the cytoplasmic contents of the mother cell are partitioned into the daughter cells, and how the daughters are positioned within the tissue. During metaphase in the early Caenorhabditis elegans embryo, the spindle is aligned and centered on the anterior-posterior axis by a microtubule-dependent machinery that exerts restoring forces when the spindle is displaced from the center. To investigate the accuracy and stability of centering, we tracked the position and orientation of the mitotic spindle during the first cell division with high temporal and spatial resolution. We found that the precision is remarkably high: the cell-to-cell variation in the transverse position of the center of the spindle during metaphase, as measured by the standard deviation, was only 1.5% of the length of the short axis of the cell. Spindle position is also very stable: the standard deviation of the fluctuations in transverse spindle position during metaphase was only 0.5% of the short axis of the cell. Assuming that stability is limited by fluctuations in the number of independent motor elements such as microtubules or dyneins underlying the centering machinery, we infer that the number is ∼1000, consistent with the several thousand of astral microtubules in these cells. Astral microtubules grow out from the two spindle poles, make contact with the cell cortex, and then shrink back shortly thereafter. The high stability of centering can be accounted for quantitatively if, while making contact with the cortex, the astral microtubules buckle as they exert compressive, pushing forces. We thus propose that the large number of microtubules in the asters provides a highly precise mechanism for positioning the spindle during metaphase while assembly is completed before the onset of anaphase.

Codon adaptation-based control of protein expression in C. elegans.

We present a method to control protein levels under native genetic regulation in Caenorhabditis elegans by using synthetic genes with adapted codons. We found that the force acting on the spindle in C. elegans embryos was related to the amount of the G-protein regulator GPR-1/2. Codon-adapted versions of any C. elegans gene can be designed using our web tool, C. elegans codon adapter.

Microtubule length correlates with spindle length in C. elegans meiosis.

The accurate segregation of chromosomes during female meiosis relies on the precise assembly and function of the meiotic spindle, a dynamic structure primarily composed of microtubules. Despite the crucial role of microtubule dynamics in this process, the relationship between microtubule length and spindle size remains elusive. Leveraging Caenorhabditis elegans as a model system, we combined electron tomography and live imaging to investigate this correlation. Our analysis revealed significant changes in spindle length throughout meiosis, coupled with alterations in microtubule length. Surprisingly, while spindle size decreases during the initial stages of anaphase, the size of antiparallel microtubule overlap decreased as well. Detailed electron tomography shows a positive correlation between microtubule length and spindle size, indicating a role of microtubule length in determining spindle dimensions. Notably, microtubule numbers displayed no significant association with spindle length, highlighting the dominance of microtubule length regulation in spindle size determination. Depletion of the microtubule depolymerase KLP-7 led to elongated metaphase spindles with increased microtubule length, supporting the link between microtubule length and spindle size. These findings underscore the pivotal role of regulating microtubule dynamics, and thus microtubule length, in governing spindle rearrangements during meiotic division, shedding light on fundamental mechanisms dictating spindle architecture.

Roles of Tubulin Concentration during Prometaphase and Ran-GTP during Anaphase of Caenorhabditis elegans Meiosis.

In many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in Caenorhabditis elegans oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF, but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes.

Roles of Tubulin Concentration during Prometaphase and Ran-GTP during Anaphase of C. elegans meiosis.

In many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in C. elegans oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes.

iNOS is necessary for GBP-mediated T. gondii clearance in murine macrophages via vacuole nitration and intravacuolar network collapse.

Toxoplasma gondii is an obligate intracellular parasite of rodents and humans. Interferon-inducible guanylate binding proteins (GBPs) are mediators of T. gondii clearance, however, this mechanism is incomplete. Here, using automated spatially targeted optical micro proteomics we demonstrate that inducible nitric oxide synthetase (iNOS) is highly enriched at GBP2+ parasitophorous vacuoles (PV) in murine macrophages. iNOS expression in macrophages is necessary to limit T. gondii load in vivo and in vitro. Although iNOS activity is dispensable for GBP2 recruitment and PV membrane ruffling; parasites can replicate, egress and shed GBP2 when iNOS is inhibited. T. gondii clearance by iNOS requires nitric oxide, leading to nitration of the PV and collapse of the intravacuolar network of membranes in a chromosome 3 GBP-dependent manner. We conclude that reactive nitrogen species generated by iNOS cooperate with GBPs to target distinct structures in the PV that are necessary for optimal parasite clearance in macrophages.

Pannexin-3 stabilizes the transcription factor Bcl6 in a channel-independent manner to protect against vascular oxidative stress.

Targeted degradation regulates the activity of the transcriptional repressor Bcl6 and its ability to suppress oxidative stress and inflammation. Here, we report that abundance of endothelial Bcl6 is determined by its interaction with Golgi-localized pannexin 3 (Panx3) and that Bcl6 transcriptional activity protects against vascular oxidative stress. Consistent with data from obese, hypertensive humans, mice with an endothelial cell-specific deficiency in Panx3 had spontaneous systemic hypertension without obvious changes in channel function, as assessed by Ca2+ handling, ATP amounts, or Golgi luminal pH. Panx3 bound to Bcl6, and its absence reduced Bcl6 protein abundance, suggesting that the interaction with Panx3 stabilized Bcl6 by preventing its degradation. Panx3 deficiency was associated with increased expression of the gene encoding the H2O2-producing enzyme Nox4, which is normally repressed by Bcl6, resulting in H2O2-induced oxidative damage in the vasculature. Catalase rescued impaired vasodilation in mice lacking endothelial Panx3. Administration of a newly developed peptide to inhibit the Panx3-Bcl6 interaction recapitulated the increase in Nox4 expression and in blood pressure seen in mice with endothelial Panx3 deficiency. Panx3-Bcl6-Nox4 dysregulation occurred in obesity-related hypertension, but not when hypertension was induced in the absence of obesity. Our findings provide insight into a channel-independent role of Panx3 wherein its interaction with Bcl6 determines vascular oxidative state, particularly under the adverse conditions of obesity.

Correlative light and electron microscopy for the analysis of cell division.

Correlative light and electron microscopy (CLEM) has recently gained increasing attention, because it enables the acquisition of dynamic as well as ultrastructural information about subcellular processes. It is the power of combining the two imaging modalities that gives additional information as compared to using the imaging techniques separately. Here, we briefly summarize two CLEM approaches for the analysis of cells in mitosis and cytokinesis.

Mitotic events depend on regulation of PLK-1 levels by the mitochondrial protein SPD-3.

In metazoans, Polo Kinase (Plk1) controls several mitotic events including nuclear envelope breakdown, centrosome maturation and kinetochore assembly. Here we show that mitotic events regulated by Polo Like Kinase (PLK-1) in early C. elegans embryos depend on the mitochondrial-localized protein SPD-3. spd-3 mutant one-cell embryos contain abnormally positioned mitotic chromosomes and prematurely and asymmetrically disassemble the nuclear lamina. Nuclear envelope breakdown (NEBD) in C. elegans requires direct dephosphorylation of lamin by PLK-1. In spd-3 mutants PLK-1 levels are ~6X higher in comparison to control embryos and PLK-1::GFP was highly accumulated at centrosomes, the nuclear envelope, nucleoplasm, and chromosomes prior to NEBD. Partial depletion of plk-1 in spd-3 mutant embryos rescued mitotic chromosome and spindle positioning defects indicating that these phenotypes result from higher PLK-1 levels and thus activity. Our data suggests that the mitochondrial SPD-3 protein controls NEBD and chromosome positioning by regulating the endogenous levels of PLK-1 during early embryogenesis in C. elegans . This finding suggests a novel link between mitochondria and mitotic events by controlling the amount of a key mitotic regulator, PLK-1 and thus may have further implications in the context of cancers or age-related diseases and infertility as it provides a novel link between mitochondria and mitosis.

Loss of the mitochondrial protein SPD-3 elevates PLK-1 levels and dysregulates mitotic events.

In metazoans, Polo-like kinase (PLK1) controls several mitotic events including nuclear envelope breakdown, centrosome maturation, spindle assembly and progression through mitosis. Here we show that a mutation in the mitochondria-localized protein SPD-3 affects mitotic events by inducing elevated levels of PLK-1 in early Caenorhabditis elegans embryos. SPD-3 mutant embryos contain abnormally positioned mitotic chromosomes, show a delay in anaphase onset and asymmetrically disassemble the nuclear lamina. We found that more PLK-1 accumulated on centrosomes, nuclear envelope, nucleoplasm, and chromatin before NEBD, suggesting that PLK-1 overexpression is responsible for some of the observed mitotic phenotypes. In agreement with this, the chromosome positioning defects of the spd-3(oj35) mutant could be rescued by reducing PLK-1 levels. Our data suggests that the mitochondrial SPD-3 protein affects chromosome positioning and nuclear envelope integrity by up-regulating the endogenous levels of PLK-1 during early embryogenesis in C. elegans This finding suggests a novel link between mitochondria and nuclear envelope dynamics and chromosome positioning by increasing the amount of a key mitotic regulator, PLK-1, providing a novel link between mitochondria and mitosis.

Mechanisms of cilia regeneration in Xenopus multiciliated epithelium in vivo.

Cilia regeneration is a physiological event, and while studied extensively in unicellular organisms, it remains poorly understood in vertebrates. In this study, using Xenopus multiciliated cells (MCCs) as a model, we demonstrate that, unlike unicellular organisms, deciliation removes the transition zone (TZ) along with the ciliary axoneme. While MCCs immediately begin the regeneration of the ciliary axoneme, surprisingly, the assembly of TZ was delayed. Instead, ciliary tip proteins, Sentan and Clamp, were the first to localize to regenerating cilia. Using cycloheximide (CHX) to block new protein synthesis, we show that the TZ protein B9d1 is not a component of the cilia precursor pool and requires new transcription/translation providing insights into the delayed repair of TZ. Moreover, CHX treatment led MCCs to assemble fewer (~ ten compared to ~150 in controls) but about wild-type length (78% of WT) cilia by gradually concentrating ciliogenesis proteins like IFT43 at a select few basal bodies, highlighting the exciting possibility of protein transport between basal bodies to facilitate faster regeneration in cells with multiple cilia. In summary, we demonstrate that MCCs begin regeneration with the assembly of ciliary tip and axoneme followed by TZ, questioning the importance of TZ in motile ciliogenesis.

Live Cell Imaging of ATP Levels Reveals Metabolic Compartmentalization within Motoneurons and Early Metabolic Changes in FUS ALS Motoneurons.

Motoneurons are one of the most energy-demanding cell types and a primary target in Amyotrophic lateral sclerosis (ALS), a debilitating and lethal neurodegenerative disorder without currently available effective treatments. Disruption of mitochondrial ultrastructure, transport, and metabolism is a commonly reported phenotype in ALS models and can critically affect survival and the proper function of motor neurons. However, how changes in metabolic rates contribute to ALS progression is not fully understood yet. Here, we utilize hiPCS-derived motoneuron cultures and live imaging quantitative techniques to evaluate metabolic rates in fused in sarcoma (FUS)-ALS model cells. We show that differentiation and maturation of motoneurons are accompanied by an overall upregulation of mitochondrial components and a significant increase in metabolic rates that correspond to their high energy-demanding state. Detailed compartment-specific live measurements using a fluorescent ATP sensor and FLIM imaging show significantly lower levels of ATP in the somas of cells carrying FUS-ALS mutations. These changes lead to the increased vulnerability of diseased motoneurons to further metabolic challenges with mitochondrial inhibitors and could be due to the disruption of mitochondrial inner membrane integrity and an increase in its proton leakage. Furthermore, our measurements demonstrate heterogeneity between axonal and somatic compartments, with lower relative levels of ATP in axons. Our observations strongly support the hypothesis that mutated FUS impacts the metabolic states of motoneurons and makes them more susceptible to further neurodegenerative mechanisms.

Lack of chromokinesin Klp-19 creates a more rigid midzone and affects force transmission during anaphase in C. elegans.

Recent studies have highlighted the significance of the spindle midzone - the region positioned between chromosomes - in ensuring proper chromosome segregation. By combining advanced 3D electron tomography and cutting-edge light microscopy we have discovered a previously unknown role of the regulation of microtubule dynamics within the spindle midzone of C. elegans. Using Fluorescence recovery after photobleaching and a combination of second harmonic generation and two-photon fluorescence microscopy, we found that the length of the antiparallel microtubule overlap zone in the spindle midzone is constant throughout anaphase, and independent of cortical pulling forces as well as the presence of the microtubule bundling protein SPD-1. Further investigations of SPD-1 and the chromokinesin KLP-19 in C. elegans suggest that KLP-19 regulates the overlap length and functions independently of SPD-1. Our data shows that KLP-19 plays an active role in regulating the length and turn-over of microtubules within the midzone as well as the size of the antiparallel overlap region throughout mitosis. Depletion of KLP-19 in mitosis leads to an increase in microtubule length in the spindle midzone, which also leads to increased microtubule - microtubule interaction, thus building up a more robust microtubule network. The spindle is globally stiffer and more stable, which has implications for the transmission of forces within the spindle affecting chromosome segregation dynamics. Our data shows that by localizing KLP-19 to the spindle midzone in anaphase microtubule dynamics can be locally controlled allowing the formation of a functional midzone.

Ndc1 drives nuclear pore complex assembly independent of membrane biogenesis to promote nuclear formation and growth.

The nuclear envelope (NE) assembles and grows from bilayer lipids produced at the endoplasmic reticulum (ER). How ER membrane incorporation coordinates with assembly of nuclear pore complexes (NPCs) to generate a functional NE is not well understood. Here, we use the stereotypical first division of the early C. elegans embryo to test the role of the membrane-associated nucleoporin Ndc1 in coupling NPC assembly to NE formation and growth. 3D-EM tomography of reforming and expanded NEs establishes that Ndc1 determines NPC density. Loss of ndc1 results in faster turnover of the outer scaffold nucleoporin Nup160 at the NE, providing an explanation for how Ndc1 controls NPC number. NE formation fails in the absence of both Ndc1 and the inner ring component Nup53, suggesting partially redundant roles in NPC assembly. Importantly, upregulation of membrane synthesis restored the slow rate of nuclear growth resulting from loss of ndc1 but not from loss of nup53. Thus, membrane biogenesis can be decoupled from Ndc1-mediated NPC assembly to promote nuclear growth. Together, our data suggest that Ndc1 functions in parallel with Nup53 and membrane biogenesis to control NPC density and nuclear size.