GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LßT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of ß-actin, with elevated levels occurring at 10 min. The level of ß-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of ß-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of ß-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LßT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.

Differential nuclear import sets the timing of protein access to the embryonic genome.

The development of a fertilized egg to an embryo requires the proper temporal control of gene expression. During cell differentiation, timing is often controlled via cascades of transcription factors (TFs). However, in early development, transcription is often inactive, and many TF levels stay constant, suggesting that alternative mechanisms govern the observed rapid and ordered onset of gene expression. Here, we find that in early embryonic development access of maternally deposited nuclear proteins to the genome is temporally ordered via importin affinities, thereby timing the expression of downstream targets. We quantify changes in the nuclear proteome during early development and find that nuclear proteins, such as TFs and RNA polymerases, enter the nucleus sequentially. Moreover, we find that the timing of nuclear proteins' access to the genome corresponds to the timing of downstream gene activation. We show that the affinity of proteins to importin is a major determinant in the timing of protein entry into embryonic nuclei. Thus, we propose a mechanism by which embryos encode the timing of gene expression in early development via biochemical affinities. This process could be critical for embryos to organize themselves before deploying the regulatory cascades that control cell identities.

Dynein-dependent collection of membranes defines the architecture and position of microtubule asters in isolated, geometrically confined volumes of cell-free extracts.

It is well established that changes in the underlying architecture of the cell's microtubule (MT) network can affect organelle organization within the cytoplasm, but it remains unclear whether the spatial arrangement of organelles reciprocally influences the MT network. Here we use a combination of cell-free extracts and hydrogel microenclosures to characterize the relationship between membranes and MTs during MT aster centration. We found that initially disperse ER membranes are collected by the aster and compacted near its nucleating center, all while the whole ensemble moves toward the geometric center of its confining enclosure. Once there, aster MTs adopt a bull's-eye pattern with a high-density annular ring of MTs surrounding the compacted membrane core of lower MT density. Formation of this pattern was inhibited when dynein-dependent transport was perturbed or when membranes were depleted from the extracts. Asters in membrane-depleted extracts were able to move away from the most proximal wall but failed to center in cylindrical enclosures with diameters greater than or equal to 150 µm. Taken as whole, our data suggest that the dynein-dependent transport of membranes buttresses MTs near the aster center and that this plays an important role in modulating aster architecture and position.

The Cytoskeleton and Its Roles in Self-Organization Phenomena: Insights from Xenopus Egg Extracts.

Self-organization of and by the cytoskeleton is central to the biology of the cell. Since their introduction in the early 1980s, cytoplasmic extracts derived from the eggs of the African clawed-frog, Xenopus laevis, have flourished as a major experimental system to study the various facets of cytoskeleton-dependent self-organization. Over the years, the many investigations that have used these extracts uniquely benefited from their simplified cell cycle, large experimental volumes, biochemical tractability and cell-free nature. Here, we review the contributions of egg extracts to our understanding of the cytoplasmic aspects of self-organization by the microtubule and the actomyosin cytoskeletons as well as the importance of cytoskeletal filaments in organizing nuclear structure and function.

Microfluidic encapsulation of Xenopus laevis cell-free extracts using hydrogel photolithography.

Cell-free extract derived from the eggs of the African clawed frog Xenopus laevis is a well-established model system that has been used historically in bulk aliquots. Here, we describe a microfluidic approach for isolating discrete, biologically relevant volumes of cell-free extract, with more expansive and precise control of extract shape compared with extract-oil emulsions. This approach is useful for investigating the mechanics of intracellular processes affected by cell geometry or cytoplasmic volume, including organelle scaling and positioning mechanisms. For complete details on the use and execution of this protocol, please refer to Geisterfer et al. (2020).

Microtubule Growth Rates Are Sensitive to Global and Local Changes in Microtubule Plus-End Density.

The microtubule cytoskeleton plays critically important roles in numerous cellular functions in eukaryotes, and it does so across a functionally diverse and morphologically disparate range of cell types [1]. In these roles, microtubule assemblies must adopt distinct morphologies and physical dimensions to perform specific functions [2-5]. As such, these macromolecular assemblies-as well as the dynamics of the individual microtubule polymers from which they are made-must scale and change in accordance with cell size, geometry, and function. Microtubules in cells typically assemble to a steady state in mass, leaving enough of their tubulin subunits soluble to allow rapid growth and turnover. This suggests some negative feedback that limits the extent of assembly, for example, decrease in growth rate, or increase in catastrophe rate, as the soluble subunit pool decreases. Although these ideas have informed the field for decades, they have not been observed experimentally. Here, we describe the application of an experimental approach that combines cell-free extracts with photo-patterned hydrogel micro-enclosures as a means to investigate microtubule dynamics in cytoplasmic volumes of defined size and shape. Our measurements reveal a negative correlation between microtubule plus-end density and microtubule growth rates and suggest that these rates are sensitive to the presence of nearby growing ends.

Instance-Level Microtubule Tracking.

We propose a new method of instance-level microtubule (MT) tracking in time-lapse image series using recurrent attention. Our novel deep learning algorithm segments individual MTs at each frame. Segmentation results from successive frames are used to assign correspondences among MTs. This ultimately generates a distinct path trajectory for each MT through the frames. Based on these trajectories, we estimate MT velocities. To validate our proposed technique, we conduct experiments using real and simulated data. We use statistics derived from real time-lapse series of MT gliding assays to simulate realistic MT time-lapse image series in our simulated data. This data set is employed as pre-training and hyperparameter optimization for our network before training on the real data. Our experimental results show that the proposed supervised learning algorithm improves the precision for MT instance velocity estimation drastically to 71.3% from the baseline result (29.3%). We also demonstrate how the inclusion of temporal information into our deep network can reduce the false negative rates from 67.8% (baseline) down to 28.7% (proposed). Our findings in this work are expected to help biologists characterize the spatial arrangement of MTs, specifically the effects of MT-MT interactions.

Nucleoplasmin is a limiting component in the scaling of nuclear size with cytoplasmic volume.

How nuclear size is regulated relative to cell size is a fundamental cell biological question. Reductions in both cell and nuclear sizes during Xenopus laevis embryogenesis provide a robust scaling system to study mechanisms of nuclear size regulation. To test if the volume of embryonic cytoplasm is limiting for nuclear growth, we encapsulated gastrula-stage embryonic cytoplasm and nuclei in droplets of defined volume using microfluidics. Nuclei grew and reached new steady-state sizes as a function of cytoplasmic volume, supporting a limiting component mechanism of nuclear size control. Through biochemical fractionation, we identified the histone chaperone nucleoplasmin (Npm2) as a putative nuclear size effector. Cellular amounts of Npm2 decrease over development, and nuclear size was sensitive to Npm2 levels both in vitro and in vivo, affecting nuclear histone levels and chromatin organization. We propose that reductions in cell volume and the amounts of limiting components, such as Npm2, contribute to developmental nuclear size scaling.

Induction of a Spindle-Assembly-Competent M Phase in Xenopus Egg Extracts.

Normal mitotic spindle assembly is a prerequisite for faithful chromosome segregation and unperturbed cell-cycle progression. Precise functioning of the spindle machinery relies on conserved architectural features, such as focused poles, chromosome alignment at the metaphase plate, and proper spindle length. These morphological requirements can be achieved only within a compositionally distinct cytoplasm that results from cell-cycle-dependent regulation of specific protein levels and specific post-translational modifications. Here, we used cell-free extracts derived from Xenopus laevis eggs to recapitulate different phases of the cell cycle in vitro and to determine which components are required to render interphase cytoplasm spindle-assembly competent in the absence of protein translation. We found that addition of a nondegradable form of the master cell-cycle regulator cyclin B1 can indeed induce some biochemical and phenomenological characteristics of mitosis, but cyclin B1 alone is insufficient and actually deleterious at high levels for normal spindle assembly. In contrast, addition of a phosphomimetic form of the Greatwall-kinase effector Arpp19 with a specific concentration of nondegradable cyclin B1 rescued spindle bipolarity but resulted in larger-than-normal bipolar spindles with a misalignment of chromosomes. Both were corrected by the addition of exogenous Xkid (Xenopus homolog of human Kid/KIF22), indicating a role for this chromokinesin in regulating spindle length. These observations suggest that, of the many components degraded at mitotic exit and then replenished during the subsequent interphase, only a few are required to induce a cell-cycle transition that produces a spindle-assembly-competent cytoplasm.

Microfluidic Encapsulation of Demembranated Sperm Nuclei in Xenopus Egg Extracts.

The cell-free nature of Xenopus egg extract makes it a uniquely tractable experimental model system. The extract, effectively unconfined cytoplasm, allows the direct and relatively straight-forward addition of purified proteins and other reagents, a characteristic that renders the system amenable to many biochemical and cell biological manipulations. Accessibility to the system also facilitates the direct physical manipulation and probing of biological structures, in turn enabling mechanical properties of intracellular assemblies and organelles, such as the mitotic spindle and nucleus, to be measured. Recently, multiphase microfluidics have been combined with Xenopus egg extracts to encapsulate discrete cytoplasmic volumes. Described here is a protocol detailing the use of multiphase microfluidic devices to encapsulate sperm nuclei within extract droplets of defined size and shape. This protocol can also be applied more generally to encapsulation of microbeads and other particles.

Isolation and Demembranation of Xenopus Sperm Nuclei.

The inherent experimental advantages of intact amphibian eggs have been exploited for several decades to advance our understanding of fundamental developmental processes and the cell cycle. Characterization of these processes at the molecular level has been greatly advanced by the use of cell-free extracts, which permit the development of biochemically tractable approaches. Demembranated Xenopus laevis sperm nuclei have been used with cell-free extracts to recapitulate cell cycle progression and to control the cell cycle state of the egg extract. This system has become an invaluable and widely used tool for studies of cell cycle regulation and many downstream events. Here, we describe a protocol, derived in part from other published protocols and modified over time, for the preparation of Xenopus sperm nuclei that can be used in a variety of in vitro assays.

Fabrication of Functional Biomaterial Microstructures by in Situ Photopolymerization and Photodegradation.

The in situ fabrication of poly(ethylene glycol) diacrylate (PEGDA) hydrogel microstructures within poly(dimethylsiloxane) (PDMS)-based microfluidic networks is a versatile technique that has enabled unique applications in biosensing, medical diagnostics, and the fundamental life sciences. Hydrogel structures have previously been patterned by the lithographic photopolymerization of PEGDA hydrogel forming solutions, a process that is confounded by oxygen-permeable PDMS. Here, we introduce an alternate PEG patterning technique that relies upon the optical sculpting of features by patterned light-induced erosion of photodegradable PEGDA deemed negative projection lithography. We quantitatively compared the hydrogel micropatterning fidelity of negative projection lithography to positive projection lithography, using traditional PEGDA photopolymerization, within PDMS devices. We found that the channel depth, the local oxygen atmosphere, and the UV exposure time dictated the size and resolution of hydrogel features formed using positive projection lithography. In contrast, negative projection lithography was observed to deliver high-resolution functional features with dimensions on the order of single micrometers enabled by its facilely controlled mechanism of feature formation that is insensitive to oxygen. Next, the utility of photodegradable PEGDA was further assessed by encapsulating or conjugating bioactive molecules within photodegradable PEG matrixes to provide a route to the formation of complex and dynamically reconfigurable chemical microenvironments. Finally, we demonstrated that negative projection lithography enabled photopatterning of multilayered microscale objects without the need for precise mask alignment. The described approach for photopatterning high-resolution photolabile hydrogel microstructures directly within PDMS microchannels could enable novel microsystems of increasing complexity and sophistication for a variety of clinical and biological applications.

Tau-based fluorescent protein fusions to visualize microtubules.

The ability to visualize cytoskeletal proteins and their dynamics in living cells has been critically important in advancing our understanding of numerous cellular processes, including actin- and microtubule (MT)-dependent phenomena such as cell motility, cell division, and mitosis. Here, we describe a novel set of fluorescent protein (FP) fusions designed specifically to visualize MTs in living systems using fluorescence microscopy. Each fusion contains a FP module linked in frame to a modified phospho-deficient version of the MT-binding domain of Tau (mTMBD). We found that expressed and purified constructs containing a single mTMBD decorated Xenopus egg extract spindles more homogenously than similar constructs containing the MT-binding domain of Ensconsin, suggesting that the binding affinity of mTMBD is minimally affected by localized signaling gradients generated during mitosis. Furthermore, MT dynamics were not grossly perturbed by the presence of Tau-based FP fusions. Interestingly, the addition of a second mTMBD to the opposite terminus of our construct caused dramatic changes to the spatial localization of probes within spindles. These results support the use of Tau-based FP fusions as minimally perturbing tools to accurately visualize MTs in living systems.

Use of Xenopus cell-free extracts to study size regulation of subcellular structures.

Striking size variations are prominent throughout biology, at the organismal, cellular, and subcellular levels. Important fundamental questions concern organelle size regulation and how organelle size is regulated relative to cell size, also known as scaling. Uncovering mechanisms of organelle size regulation will inform the functional significance of size as well as the implications of misregulated size, for instance in the case of nuclear enlargement in cancer. Xenopus egg and embryo extracts are powerful cell-free systems that have been utilized extensively for mechanistic and functional studies of various organelles and subcellular structures. The open biochemical nature of the extract permits facile manipulation of its composition, and in recent years extract approaches have illuminated mechanisms of organelle size regulation. This review largely focuses on in vitro Xenopus studies that have identified regulators of nuclear and spindle size. We also discuss potential relationships between size scaling of the nucleus and spindle, size regulation of other subcellular structures, and extract experiments that have clarified developmental timing mechanisms. We conclude by offering some future prospects, notably the integration of Xenopus extract with microfluidic technology.

Nanoparticle Targeting and Cholesterol Flux Through Scavenger Receptor Type B-1 Inhibits Cellular Exosome Uptake.

Exosomes are nanoscale vesicles that mediate intercellular communication. Cellular exosome uptake mechanisms are not well defined partly due to the lack of specific inhibitors of this complex cellular process. Exosome uptake depends on cholesterol-rich membrane microdomains called lipid rafts, and can be blocked by non-specific depletion of plasma membrane cholesterol. Scavenger receptor type B-1 (SR-B1), found in lipid rafts, is a receptor for cholesterol-rich high-density lipoproteins (HDL). We hypothesized that a synthetic nanoparticle mimic of HDL (HDL NP) that binds SR-B1 and removes cholesterol through this receptor would inhibit cellular exosome uptake. In cell models, our data show that HDL NPs bind SR-B1, activate cholesterol efflux, and attenuate the influx of esterified cholesterol. As a result, HDL NP treatment results in decreased dynamics and clustering of SR-B1 contained in lipid rafts and potently inhibits cellular exosome uptake. Thus, SR-B1 and targeted HDL NPs provide a fundamental advance in studying cholesterol-dependent cellular uptake mechanisms.

Spatially segregated transcription and translation in cells of the endomembrane-containing bacterium Gemmata obscuriglobus.

The dogma of coupled transcription and translation in bacteria has been challenged by recent reports of spatial segregation of these processes within the relatively simple cellular organization of the model organisms Escherichia coli and Bacillus subtilis. The bacterial species Gemmata obscuriglobus possesses an extensive endomembrane system. The membranes generate a very convoluted intracellular architecture in which some of the cell's ribosomes appear to have less direct access to the cell's nucleoid(s) than others. This observation prompted us to test the hypothesis that a substantial proportion of G. obscuriglobus translation may be spatially segregated from transcription. Using immunofluorescence and immunoelectron microscopy, we showed that translating ribosomes are localized throughout the cell, with a quantitatively greater proportion found in regions distal to nucleoid(s). Our results extend information about the phylogenetic and morphological diversity of bacteria in which the spatial organization of transcription and translation has been studied. These findings also suggest that endomembranes may provide an obstacle to colocated transcription and translation, a role for endomembranes that has not been reported previously for a prokaryotic organism. Our studies of G. obscuriglobus may provide a useful background for consideration of the evolutionary development of eukaryotic cellular complexity and how it led to decoupled processes of gene expression in eukaryotes.

Meeting report: mitosis and nuclear structure.

The Company of Biologists Workshop entitled 'Mitosis and Nuclear Structure' was held at Wiston House, West Sussex in June 2013. It provided a unique and timely opportunity for leading experts from different fields to discuss not only their own work but also its broader context. Here we present the proceedings of this meeting and several major themes that emerged from the crosstalk between the two, as it turns out, not so disparate fields of mitosis and nuclear structure. Co-chaired by Katherine Wilson (Johns Hopkins School of Medicine, Baltimore, MD), Timothy Mitchison (Harvard University, Cambridge, MA) and Michael Rout (Rockefeller University, New York, NY), this workshop brought together a small group of scientists from a range of disciplines to discuss recent advances and connections between the areas of mitosis and nuclear structure research. Several early-career researchers (students, postdoctoral researchers, junior faculty) participated along with 20 senior scientists, including the venerable and affable Nobel Laureate Tim Hunt. Participants were encouraged to embrace unconventional thinking in the 'scientific sandbox' created by this unusual combination of researchers in the inspiring, isolated setting of the 16th-century Wiston House.

Directly probing the mechanical properties of the spindle and its matrix.

Several recent models for spindle length regulation propose an elastic pole to pole spindle matrix that is sufficiently strong to bear or antagonize forces generated by microtubules and microtubule motors. We tested this hypothesis using microneedles to skewer metaphase spindles in Xenopus laevis egg extracts. Microneedle tips inserted into a spindle just outside the metaphase plate resulted in spindle movement along the interpolar axis at a velocity slightly slower than microtubule poleward flux, bringing the nearest pole toward the needle. Spindle velocity decreased near the pole, which often split apart slowly, eventually letting the spindle move completely off the needle. When two needles were inserted on either side of the metaphase plate and rapidly moved apart, there was minimal spindle deformation until they reached the poles. In contrast, needle separation in the equatorial direction rapidly increased spindle width as constant length spindle fibers pulled the poles together. These observations indicate that an isotropic spindle matrix does not make a significant mechanical contribution to metaphase spindle length determination.

Microtubule motors in eukaryotic spindle assembly and maintenance.

The spindle is a microtubule-based structure that facilitates chromosome segregation during mitosis and meiosis. Spindle assembly from dynamic microtubule building blocks is a major challenge for the dividing cell and a process that critically requires microtubule motors. In this review we focus on the mechanisms by which microtubule motors shape the spindle. Specifically, we address how motors are thought to move and arrange microtubules to form the characteristic bipolar morphology shared by all eukaryotic spindles as well as motor-dependent mechanisms of microtubule length regulation.

Data harvesting from fields of spindles.

The mitotic spindle is essential for chromosome segregation and must be large enough to accommodate all of the chromatin in the dividing cell. In this issue, Dinarina et al. (2009) grow "fields" of spindles on coverslips to investigate the relationship between chromatin and spindle size as well as intrinsic mechanisms of spindle assembly.