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Clinical use of pancreatic β islets for regenerative medicine applications requires mass production of functional cells. Current technologies are insufficient for large-scale production in a cost-efficient manner. Here, we evaluate advantages of a porous cellulose scaffold and demonstrate scale-up to a wicking matrix bioreactor as a platform for culture of human endocrine cells. Scaffold modifications were evaluated in a multiwell platform to find the optimum surface condition for pancreatic cell expansion followed by bioreactor culture to confirm suitability. Preceding scale-up, cell morphology, viability, and proliferation of primary pancreatic cells were evaluated. Two optimal surface modifications were chosen and evaluated further for insulin secretion, cell morphology, and viable cell density for human-induced pluripotent stem cell-derived pancreatic cells at different stages of differentiation. Scale-up was accomplished with uncoated, amine-modified cellulose in a miniature bioreactor, and insulin secretion and cell metabolic profiles were determined for 13 days. We achieved 10-fold cell expansion in the bioreactor along with a significant increase in insulin secretion compared with cultures on tissue culture plastic. Our findings define a new method for expansion of pancreatic cells a on wicking matrix cellulose platform to advance cell therapy biomanufacturing for diabetes.

Neural cell interventions in spinal cord injury (SCI) have focused predominantly on transplanted multipotent neural stem/progenitor cells (NSPCs) for animal research and clinical use due to limited information on survival of spinal neurons. However, transplanted NSPC fate is unpredictable and largely governed by injury-derived matrix and cytokine factors that are often gliogenic and inflammatory. Here, using a rat cervical hemicontusion model, we evaluate the survival and integration of hiPSC-derived spinal motor neurons (SMNs) and oligodendrocyte progenitor cells (OPCs). SMNs and OPCs were differentiated in vitro through a neuromesodermal progenitor stage to mimic the natural origin of the spinal cord. We demonstrate robust survival and engraftment without additional injury site modifiers or neuroprotective biomaterials. Ex vivo differentiated neurons achieve cervical spinal cord matched transcriptomic and proteomic profiles, meeting functional electrophysiology parameters prior to transplantation. These data establish an approach for ex vivo developmentally accurate neuronal fate specification and subsequent transplantation for a more streamlined and predictable outcome in neural cell-based therapies of SCI.

The inaccessibility of scientific discovery to human development at post-implantation, including specialization and differentiation, necessitates sophisticated heterogenous 3D in vitro models. This protocol describes the generation and self-organization of human elongating multi-lineage organized (EMLO) gastruloids in suspension without the need for supplied extracellular matrix (ECM). EMLO gastruloids can be matured to large organoid states comprised of interconnected tissue microstructures of the trunk. These include correlates of the neuromesodermal progenitor-derived central nervous system, the neural crest cell-derived peripheral nervous system, the endoderm-derived primitive gut tube, and the mesoderm-derived splanchnic mesenchyme. Herein, we provide critical details for the reproducible generation of human EMLOs in vitro.

The ability to reliably repair spinal cord injuries (SCI) will be one of the greatest human achievements realized in regenerative medicine. Until recently, the cellular path to this goal has been challenging. However, as detailed developmental principles are revealed in mouse and human models, their application in the stem cell community brings trunk and spine embryology into efforts to advance human regenerative medicine. New models of posterior embryo development identify neuromesodermal progenitors (NMPs) as a major bifurcation point in generating the spinal cord and somites and is leading to production of cell types with the full range of axial identities critical for repair of trunk and spine disorders. This is coupled with organoid technologies including assembloids, circuitoids, and gastruloids. We describe a paradigm for applying developmental principles towards the goal of cell-based restorative therapies to enable reproducible and effective near-term clinical interventions.

Summary To repair neural circuitry following spinal cord injury (SCI), neural stem cell (NSC) transplantation has held a primary focus; however, stochastic outcomes generate challenges driven in part by NSC differentiation and tumor formation. The recent ability to generate regionally specific neurons and their support cells now allows consideration of directed therapeutic approaches with pre-differentiated and networked spinal neural cells. Here, we form encapsulated, transplantable neuronal networks of regionally matched cervical spinal motor neurons, interneurons, and oligodendrocyte progenitor cells derived through trunk-biased neuromesodermal progenitors. We direct neurite formation in alginate-based neural ribbons to generate electrically active, synaptically connected networks, characterized by electrophysiology and calcium imaging before transplantation into rodent models of contused SCI for evaluation at 10-day and 6-week timepoints. The in vivo analyses demonstrate viability and retention of interconnected synaptic networks that readily integrate with the host parenchyma to advance goals of transplantable neural circuitry for SCI treatment., Graphical abstract, Highlights • Neuromesodermal progenitor derivation of human spinal neurons as therapeutic cells • Neural ribbons bridge in vitro network formation and in vivo host transplantation • In vivo visualization of encapsulated graft placement with magnetic resonance imaging • Six-week viability of human neuronal networks with OPCs in rat contusion SCI, Neuroscience; Bioengineering; Tissue engineering

Human stem cell technologies including self-assembling 3D tissue models provide unprecedented access to early neurodevelopment and enable fundamental insights into neuropathologies. Gastruloid models have yet to be used to investigate the developing nervous system. Here we generate elongating multi-lineage-organized (EMLO) gastruloids with trunk identity that co-develop central and peripheral nervous system (CNS, PNS) correlates. We track migrating neural crest cells that differentiate to form peripheral neurons integrated with an upstream spinal cord region. This follows initial EMLO polarization events, and is coordinated with primitive gut tube elongation and cardiomyocyte differentiation. By immunofluorescence of multi-lineage and functional biomarkers, we evaluate EMLOs over a twenty-two day period, and apply them to investigate the impact of mu opioid receptor modulation on neuronal activity. This comprehensive study demonstrates a novel combined CNS-PNS model of early organogenesis and integration events in the trunk to benefit human biomedical research.

Stem cell technologies including self-assembling 3D tissue models provide access to early human neurodevelopment and fundamental insights into neuropathologies. Gastruloid models have not been used to investigate co-developing central and peripheral neuronal systems with trunk mesendoderm which we achieve here in elongating multi-lineage organized (EMLO) gastruloids. We evaluate EMLOs over a forty-day period, applying immunofluorescence of multi-lineage and functional biomarkers, including day 16 single-cell RNA-Seq, and evaluation of ectodermal and non-ectodermal neural crest cells (NCCs). We identify NCCs that differentiate to form peripheral neurons integrated with an upstream spinal cord region after day 8. This follows initial EMLO polarization events that coordinate with endoderm differentiation and primitive gut tube formation during multicellular spatial reorganization. This combined human central-peripheral nervous system model of early organogenesis highlights developmental events of mesendoderm and neuromuscular trunk regions and enables systemic studies of tissue interactions and innervation of neuromuscular, enteric and cardiac relevance., The authors generate EMLOs (elongating multi-lineage organized gastruloids): organoids that self-organize to form compartments with characteristics of the central nervous system, peripheral nervous system, mesenchyme, and gut tube.

Improved human stem cell interventions to treat CNS trauma requires continued expansion of in vitro models and delivery platforms to fill gaps in analysis and treatment. Transplanted neural stem cells (NSCs) face unique, multi-faceted challenges beyond survival that include differentiation, maturation, and integration into a complex cytokine-releasing microenvironment that impinges on a multipotent cell type. Alternate strategies to transplant neurons and neuronal networks deserve reevaluation, particularly since novel differentiation protocols mimicking region-specific developmental and positional cues have recently emerged. To investigate transplantation of neurons and their early networks, we generate in vitro neural ribbons containing spinal neurons and support cells anatomically matched for cervical spinal cord injury (SCI). These glutamate-responsive, electrically-active neural ribbons apply a new hiPSC differentiation strategy transiting through neuromesodermal progenitors (NMps) to derive developmentally relevant spinal motor neurons (SMNs), interneurons (INs), and oligodendrocyte progenitor cells (OPCs). Bioinformatic profiling validates region-specific identities. Neurons and neuronal networks are functionally evaluated for action potential firing, calcium signaling, population activity, and synaptogenesis. NMp-derived neurons survive in vivo within the subacute phase hemi-contusion injury cavity when delivered either as free suspension or as encapsulated networks of pre-formed CNS cytoarchitectures. Delivery as encapsulated networks further supports survival of lower cell numbers and rapid graft penetration into host tissue. Neural network ribbons therefore provide a novel intermediary approach between cell suspensions and complex organoids for investigating network formation and early transplantation events with hiPSC-derived neurons, providing flexibility to rapidly tune cell type(s), cell ratios, and traceable biomarkers.Significance StatementIn the two decades since human stem cell technologies have emerged, the challenge has remained to improve the developmentally relevant derivation of therapeutic cells. The ability to now generate anatomically matched neurons for SCI necessitates a re-evaluation of these cells and their networks in vitro and in vivo. In this study, we apply developmental cues via neuromesodermal progenitors to generate spinal neurons from hiPSCs. Genetic and functional evaluation of these cells as in vitro neuronal networks, due to their capacity to survive and graft effectively within the rat subacute contusion cavity, offer novel approaches for customizing SCI transplantation. This work demonstrates a strategy to develop transplantable, chemically-responsive networks linking in vitro models with injury customization towards improved in vivo outcomes.

Multipotent neural stem cells (NSCs) are widely applied in pre-clinical and clinical trials as a cell source to promote tissue regeneration in neurodegenerative diseases. Frequently delivered as dissociated cells, aggregates or self-organized rosettes, it is unknown whether disruption of the NSC rosette morphology or method of formation affect signaling profiles of these cells that may impact uniformity of outcomes in cell therapies. Here we generate a neural cell-cell interaction microchip (NCCIM) as an in vitro platform to simultaneously track an informed panel of cytokines and co-evaluate cell morphology and biomarker expression coupled to a sandwich ELISA platform. We apply multiplex in situ tagging technology (MIST) to evaluate ten cytokines (PDGF-AA, GDNF, BDNF, IGF-1, FGF-2, IL-6, BMP-4, CNTF, β-NGF, NT-3) on microchips for EB-derived rosettes, single cell dissociated rosettes and reformed rosette neurospheres. Of the cytokines evaluated, EB-derived rosettes secrete PDGF-AA, GDNF and FGF-2 prominently, whereas this profile is temporarily lost upon dissociation to single cells and in reformed neurospheres two additional cytokines, BDNF and β-NGF, are also secreted. This study on NSC rosettes demonstrates the development, versatility and utility of the NCCIM as a sensitive multiplex detector of cytokine signaling in a high throughput and controlled microenvironment. The NCCIM is expected to provide important new information to refine cell source choices in therapies as well as to support development of informative 2D or 3D in vitro models including areas of neurodegeneration or neuroplasticity.

Cell therapy for the injured spinal cord will rely on combined advances in human stem cell technologies and delivery strategies. Here we encapsulate homotypic spinal cord neural stem cells (scNSCs) in an alginate-based neural ribbon delivery platform. We perform a comprehensive in vitro analysis and qualitatively demonstrate graft survival and injury site retention using a rat C4 hemi-contusion model. Pre-configured neural ribbons are transport-stable modules that enable site-ready injection, and can support scNSC survival and retention in vivo. Neural ribbons offer multifunctionality in vitro including co-encapsulation of the injury site extracellular matrix modifier chondroitinase ABC (chABC), tested here in glial scar models, and ability of cervically-patterned scNSCs to differentiate within neural ribbons and project axons for integration with 3-D external matrices. This is the first extensive in vitro characterization of neural ribbon technology, and constitutes a plausible method for reproducible delivery, placement, and retention of viable neural cells in vivo.

Arunima Banerjee,1 Janet L Paluh,2 Amitava Mukherjee,3 K Gaurav Kumar,1 Archisman Ghosh,1 Mrinal K Naskar1 1Department of Electronics and Tele-Communication Engineering, Jadavpur University, Kolkata, India; 2College of Nanoscale Science, Nanobioscience Constellation, State University of New York Polytechnic Institute, Albany, NY, USA; 3Globsyn Business School, Kolkata, India Aim: In tauopathies such as Alzheimer’s disease (AD), molecular changes spanning multiple subcellular compartments of the neuron contribute to neurodegeneration and altered axonal signaling. Computational modeling of end-to-end linked events benefit mechanistic analysis and can be informative to understand disease progression and accelerate development of effective therapies. In the calcium-amyloid beta model of AD, calcium ions that are an important regulator of neuronal function undergo dysregulated homeostasis that disrupts cargo loading for neurotrophic signaling along axonal microtubules (MTs). The aim of the present study was to develop a computational model of the neuron using a layered architecture simulation that enables us to evaluate the functionalities of several interlinked components in the calcium-amyloid beta model. Methods: The elevation of intracellular calcium levels is modeled upon binding of amyloid beta oligomers (AβOs) to calcium channels or as a result of membrane insertion of oligomeric Aβ1-42 to form pores/channels. The resulting subsequent Ca2+ disruption of dense core vesicle (DCV)-kinesin cargo loading and transport of brain-derived neurotrophic factor (BDNF) on axonal MTs are then evaluated. Our model applies published experimental data on calcium channel manipulation of DCV-BDNF and incorporates organizational complexity of the axon as bundled polar and discontinuous MTs. The interoperability simulation is based on the Institute of Electrical and Electronics Engineers standard association P1906.1 framework for nanoscale and molecular communication. Results: Our analysis provides new spatiotemporal insights into the end-to-end signaling events linking calcium dysregulation and BDNF transport and by simulation compares the relative impact of dysregulation of calcium levels by AβO-channel interactions, oligomeric Aβ1-42 pores/channel formation, and release of calcium by internal stores. The flexible platform of our model allows continued expansion of molecular details including mechanistic and morphological parameters of axonal cytoskeleton networks as they become available to test disease and treatment predictions. Conclusion: The present model will benefit future drug studies on calcium homeostasis and dysregulation linked to measurable neural functional outcomes. The algorithms used can also link to other multiscale multi-cellular modeling platforms to fill in molecular gaps that we believe will assist in broadening and refining multiscale computational maps of neurodegeneration. Keywords: amyloid beta oligomers, AβO pore, calcium hypothesis Alzheimer’s disease, BDNF, kinesin, axonal transport, nanocommunication, simulation

As a renewable source of progenitor and mature cell types, human iPSCs offer promise to replace or restore impaired neural circuits in the injured nervous system. CNS delivery of neural stem cell suspensions lacking pre-connected networks requires their differentiation, maturation, and connectivity in vivo. By contrast, integrating specified neural networks and their support cells with biomaterials allows the advancement of tailored transplantation strategies. Here, we demonstrate encapsulated 3D electrically-active and glutamate-responsive neural ribbon networks using differentiated hiPSC-derived spinal motor neurons and oligodendrocyte progenitor support cells. We apply comprehensive multi-parameter analysis in vitro to interrogate synaptogenesis, myotube innervation, action potential firing and relevant bioinformatics profiles. As proof-of-principle for spinal cord delivery, we inject neural ribbons within the contused injury cavity in rat and qualitatively demonstrate cell viability. Neural ribbons therefore constitute a tunable method to deliver pre-configured, physiologically-matched functional cytoarchitectures to the CNS.

In pluripotent stem cell differentiation, embryoid bodies (EBs) provide a three-dimensional [3D] multicellular precursor in lineage specification. The internal structure of EBs is not well characterized yet is predicted to be an important parameter to differentiation. Here, we use custom SU-8 molds to generate transparent lithography-templated arrays of polydimethylsiloxane (LTA-PDMS) for high throughput analysis of human embryonic stem cell (hESC) EB formation and internal architecture. EBs formed in 200 and 500 μm diameter microarray wells by use of single cells, 2D clusters, or 3D early aggregates were compared. We observe that 200 μm EBs are monocystic versus 500 μm multicystic EBs that contain macro, meso and microsized cysts. In adherent differentiation of 500 μm EBs, the multicystic character impairs the 3D to 2D transition creating non-uniform monolayers. Our findings reveal that EB core structure has a size-dependent character that influences its architecture and cell population uniformity during early differentiation.

We report a novel strategy for fabrication of multifunctional dumbbell particles (DPs) through click chemistry for monitoring single-cell cytokine releasing. Two different types of DPs were prepared on a large scale through covalent bioorthogonal reaction between methyltetrazine and trans-cyclooctene on a microchip under a magnetic field. After collection of the DPs, the two sides of each particle were further functionalized with different antibodies for cell capturing and cytokine detection, respectively. These DPs labeled with different fluorescent dyes have been used for multiplex detection and analysis of cytokines secreted by single live cells. Our results show that this new type of DPs are promising for applications in cell sorting, bioimaging, single-cell analysis, and biomedical diagnostics.

Summary Microtubule-organizing centers such as the γ-tubulin ring complex (γ-TuRC) act as a template for polarized growth and regulation of microtubules that are essential for diverse cellular structures and processes in eukaryotes. New structural models of the budding yeast γ-tubulin small complex (γ-TuSC) of the γ-TuRC combined with functional studies done in multiple eukaryotes are revealing the first mechanistic clues into control of microtubule nucleation and organization. Cross-species studies of human and budding yeast γ-TuSC proteins in fission yeast revealed conserved and divergent structural and functional features of the γ-TuSC. We show genetically that GCP3/Spc98 function is fully conserved with Alp6 across species but that functional differences exist between GCP2/Spc97 and Alp4. By further analysis of human γ-TuSC proteins, we found that GCP3 assembles normally into the >2000 kDa fission yeast γ-TuRC and that the GCP3 gene replaces fission yeast alp6. Interestingly, human GCP2 replaces the essential alp4 gene but is unable to rescue a normally recessive G1 defect of the alp4-1891 allele that results in loss of γ-TuRC from poles in subsequent cell cycles. Biochemically, GCP2 incorporation into fission yeast γ-TuRC is limited in the presence of Alp4; instead, the bulk of GCP2 fractionates as smaller complexes. By generating a functional Alp4–GCP2 chimeric protein we determined that the GCP2 N-terminal domain limits its ability to fully displace or compete with Alp4 during γ-TuRC assembly. Our findings have broad importance for understanding the essential domains of γ-TuSC proteins in the γ-TuRC mechanism.

The realization of personalized medicine through human induced pluripotent stem cell (iPSC) technology can be advanced by transcriptomics, epigenomics, and bioinformatics that inform on genetic pathways directing tissue development and function. When possible, population diversity should be included in new studies as resources become available. Previously we derived replicate iPSC lines of African American, Hispanic-Latino and Asian self-designated ethnically diverse (ED) origins with normal karyotype, verified teratoma formation, pluripotency biomarkers, and tri-lineage in vitro commitment. Here we perform bioinformatics of RNA-Seq and ChIP-seq pluripotency data sets for two replicate Asian and Hispanic-Latino ED-iPSC lines that reveal differences in generation of contractile cardiomyocytes but similar and robust differentiation to multiple neural, pancreatic, and smooth muscle cell types. We identify shared and distinct genes and contributing pathways in the replicate ED-iPSC lines to enhance our ability to understand how reprogramming to iPSC impacts genes and pathways contributing to cardiomyocyte contractility potential.

Kinesin-like proteins are integral to formation and function of a conserved mitotic spindle apparatus that directs chromosome segregation and precedes cell division. Ubiquitous to the mechanism of spindle assembly and stability are balanced Kinesin-5 promoting and Kinesin-14 opposing forces. Distinct Kinesin-14 roles in bipolarity in eukaryotes have not been shown, but are suggested by gamma-tubulin-based pole interactions that affect establishment and by microtubule cross-linking and sliding that maintain bipolarity and spindle length. Distinct roles also imply specialized functional domains. By cross-species analysis of compatible mechanisms in establishing mitotic bipolarity we demonstrate that Kinesin-14 human HSET (HsHSET) functionally replaces Schizosaccharomyces pombe Pkl1 and its action is similarly blocked by mutation in a Kinesin-14 binding site on gamma-tubulin. Drosophila DmNcd localizes preferentially to bundled interpolar microtubules in fission yeast and does not replace SpPkl1. Analysis of twenty-six Kinesin-14 derivatives, including Tail, Stalk or Neck-Motor chimeras, for spindle localization, spindle assembly and mitotic progression defined critical domains. The Tail of SpPkl1 contains functional elements enabling its role in spindle assembly that are distinct from but transferable to DmNcd, whereas HsHSET function utilizes both Tail and Stalk features. Our analysis is the first to demonstrate distinct mechanisms between SpPkl1 and DmNcd, and reveal that HsHSET shares functional overlap in spindle pole mechanisms.

The human genome with all its ethnic variations contributes to differences in human development, aging, disease, repair and response to medical treatments and is an exciting area of research and clinical study. The availability of well-characterized ethnically diverse stem cell lines is limited and has not kept pace with other advances in stem cell research. Here we derived xenofree ethnically diverse-human induced pluripotent stem cell (ED-iPSC) lines from fibroblasts obtained from individuals of African American, Hispanic-Latino, Asian and Caucasian ethnic origin and have characterized the lines under a uniform platform for comparative analysis. Derived ED-iPSC lines are low passage number and evaluated in vivo by teratoma formation and in vitro by high throughput microarray analysis of EB formation and early differentiation for tri-lineage commitment to endoderm, ectoderm and mesoderm. These new xenofree ED-iPSC lines represent a well-characterized valuable resource with potential for use in future research in drug discovery or clinical investigations.

The microtubule cytoskeleton is involved in regulation of cell morphology, differentiation, and cell cycle progression. Precisely controlled dynamic properties are required for these microtubule functions. To better understand how tubulin's dynamics are embedded in its primary sequence, we investigated in vivo the consequences of altering a single, highly conserved residue in β-tubulin that lies at the interface between two structural domains. The residue differs between the cold-adapted Antarctic fish and temperate animals in a manner that suggests a role in microtubule stability. Fungi, like the Antarctic fish, have a phenylalanine in this position, whereas essentially all other animals have tyrosine. We mutated the corresponding residue in fission yeast to tyrosine. Temperature effects were subtle, but time-lapse microscopy of microtubule dynamics revealed reduced depolymerization rates and increased stability. Mitotic exit signaled by breakdown of the mitotic spindle was delayed. In meiosis, microtubules displayed prolonged contact to the cell cortex during horsetail movement, followed by completion of meiosis I but frequent asymmetric failure of meiosis II spindle formation. Our results indicate that depolymerization dynamics modulated through interdomain motion may be important for regulating a subset of plus-end microtubule complexes in Schizosaccharomyces pombe.

Bacterial pyrogens, capable of penetrating dialyzer membranes, are responsible for a systemic inflammatory reaction in hemodialysis patients. Dialyzer reuse, involving rinsing of the dialyzer with pyrogen-containing water, may exacerbate this situation. Studies of the mechanism of action of endotoxin suggest that it irreversibly damages the vascular endothelium. The novel endotoxin removal method described here, is based on affinity-binding of endotoxin by the adsorbent ClarEtoxTM, a USP Class VI-certified resin that is the active component of the medical device DialGuardTM. Under standard hemodialysis operating conditions, challenge of DialGuard with Pseudomonas maltophilia supernatant-spiked dialysate, containing 35–193 EU/ml endotoxin, resulted in endotoxin levels below 0.05 EU/ml in the treated dialysate. DialGuard was able to decrease endotoxin concentrations in the dialysate from a range of 2.39–8.49 to

In pluripotent stem cell differentiation, embryoid bodies (EBs) provide a three-dimensional [3D] multicellular precursor in lineage specification. The internal structure of EBs is not well characterized yet is predicted to be an important parameter to differentiation. Here, we use custom SU-8 molds to generate transparent lithography-templated arrays of polydimethylsiloxane (LTA-PDMS) for high throughput analysis of human embryonic stem cell (hESC) EB formation and internal architecture. EBs formed in 200 and 500 μm diameter microarray wells by use of single cells, 2D clusters, or 3D early aggregates were compared. We observe that 200 μm EBs are monocystic versus 500 μm multicystic EBs that contain macro, meso and microsized cysts. In adherent differentiation of 500 μm EBs, the multicystic character impairs the 3D to 2D transition creating non-uniform monolayers. Our findings reveal that EB core structure has a size-dependent character that influences its architecture and cell population uniformity during early differentiation.

Bipolar spindle assembly is a critical control point for initiation of mitosis through nucleation and organization of spindle microtubules and is regulated by kinesin-like proteins. In fission yeast, the kinesin-14 Pkl1 binds the γ-tubulin ring complex (γ-TuRC) microtubule-organizing centre at spindle poles and can alter its structure and function. Here we show that kinesin-14 blocks microtubule nucleation in yeast and reveal that this inhibition is countered by the kinesin-5 protein, Cut7. Furthermore, we demonstrate that Cut7 binding to γ-TuRC and the Cut7 BimC domain are both required for inhibition of Pkl1. We also demonstrate that a yeast kinesin-14 peptide blocks microtubule nucleation in two human breast cancer cell lines, suggesting that this mechanism is evolutionarily conserved. In conclusion, using genetic, biochemical and cell biology approaches we uncover antagonistic control of microtubule nucleation at γ-TuRC by two kinesin-like proteins, which may represent an attractive anti-mitotic target for cancer therapies., Mitotic spindle assembly requires strict control of microtubule nucleation by γ-tubulin ring complexes. Olmsted et al. report that the kinesin-like proteins Pkl1 and Cut7 antagonistically regulate nucleation in fission yeast, and show that a Pkl1 peptide blocks spindle assembly in human cancer cells.

Mitotic spindle assembly is a critical control point in eukaryotic chromosome segregation. In fission yeast conserved mitotic kinesin-like proteins (Klps) Kinesin-14 and Kinesin-5 are emerging as novel regulators of the γ-tubulin ring complex (γ-TuRC) MTOC. While these Klps typically participate in microtubule roles for spindle assembly, alternatively in fission yeast they localize to γ-TuRC and when co-deleted a bipolar spindle forms. Our analysis by genetics, biochemistry, cell biology and nanotechnology provides new knowledge on mitotic mechanisms in S. pombe. Previous work from our lab and others uncovered a novel functional relationship between Kinesin-14 Pkl1 and all proteins of the γ-tubulin small complex (γ-TuSC). We demonstrated that Pkl1 binds γ-TuRC through Motor and Tail interactions to directly regulate its function. A Pkl1 Tail peptide is sufficient in vitro and disrupts γ-TuRC structure by removal of γ-tubulin subunits (Cell Cycle 2013), an action that is reversible. New data from our lab indicates that the essential Kinesin-5 Cut7 is actually dispensable in the absence of Pkl1 with which it shares an important antagonistic MTOC regulatory relationship. Cut-7 associates with FPLC purified γ-TuRC and binds to γ-tubulin similarly as Pkl1. We demonstrate in vivo that Pkl1 blocks microtubule nucleation from γ-TuRC and additionally influences spindle microtubule numbers and microtubule overlap. Kinetochore-based microtubule motors Klp5 and Klp6 may facilitate spindle organization, since triple mutants of Pkl1, Cut7 and either Kinesin-6 Klp9 or Kinesin-14 Klp2 are viable. Previously we showed in vivo that human Kinesin-14 HSET, but not Drosophila Ncd, functionally replaces Pkl1 and more recently that human γ-TuSC proteins GCP2 and GCP3 replace fission yeast Alp4 and Alp6 (JCS 2013). Our research highlights conserved in vivo functions of γ-TuSC and provides a new model of spindle assembly in S. pombe.

Reverse genetics in fission yeast is hindered by the lack of a versatile established plasmid shuffle system. In order to screen efficiently and accurately through plasmid-borne mutations in the essential gene for the RNA component of RNase MRP, mrp1, we have developed a system for plasmid shuffling in fission yeast using counterselection on canavanine. The system takes advantage of the ability of the Saccharomyces cerevisiae CAN1 gene to complement a Schizosaccharomyces pombe can1-1 mutation. Two general use plasmids were constructed that allow directional cloning and initial selection for histidine before counterselection by canavanine. The strain constructed for plasmid shuffling carries auxotrophic markers for ade6, leul, ura4 and his3 along with the can1-1 mutation. Using this system we examined several partial deletions and point mutations in conserved nucleotides of Schizosaccharomyces pombe RNase MRP RNA for their ability to complement a chromosomal deletion of the mrp1 gene. The degree of background canavanine resistance as well as plasmid-plasmid recombination encountered in these experiments was sufficiently low to suggest that the system we have set up for counterselection by canavanine in fission yeast using multicopy plasmids will be widely useful.

The essential gene for RNase MRP RNA, mrp1, was identified previously in Schizosaccharomyces pombe by homology to mammalian RNase MRP RNAs. Here we describe distinct site-specific mutations in RNase MRP RNA that support a conserved role for this ribonucleoprotein in nucleolar 5.8S rRNA processing. One characterized mutation, mrp1-ND90, displays dominance and results in accumulation of unspliced precursor RNAs of dimeric tRNA(Ser)-tRNA(Met)i, suggesting a novel nuclear role for RNase MRP in tRNA processing. Cells carrying the mrp1-ND90 mutation, in the absence of a wild-type copy of mrp1, additionally require the mitochondrially associated nuclear mutation ptp1-1 for viability. Analysis of this mrp1 mutation reinforces previous biochemical evidence suggesting a role for RNase MRP in mitochondrial DNA replication. Several mutations in mrp1 result in unusual cellular morphology, including alterated nuclear organization, and are consistent with a broader nuclear role for RNase MRP in regulating a nuclear signal for septation; these results are a further indication of the multifunctional nature of this ribonucleoprotein.

The γ-tubulin ring complex (γ-TuRC) is a key part of microtubule-organizing centers (MTOCs) that control microtubule polarity, organization and dynamics in eukaryotes. Understanding regulatory mechanisms of γ-TuRC function is of fundamental importance, as this complex is central to many cellular processes, including chromosome segregation, fertility, neural development, T-cell cytotoxicity and respiration. The fission yeast microtubule motor kinesin-14 Pkl1 regulates mitosis by binding to the γ-tubulin small complex (γ-TuSC), a subunit of γ-TuRC. Here we investigate the binding mechanism of Pkl1 to γ-TuSC and its functional consequences using genetics, biochemistry, peptide assays and cell biology approaches in vivo and in vitro. We identify two critical elements in the Tail domain of Pkl1 that mediate γ-TuSC binding and trigger release of γ-tubulin from γ-TuRC. Such action disrupts the MTOC and results in failed mitotic spindle assembly. This study is the first demonstration that a motor protein directly affects the structural composition of the γ-TuRC, and we provide details of this mechanism that may be of broad biological importance.

Pluripotent and multipotent cells become increasingly lineage restricted through differentiation. Alterations to the cellular proteoglycan composition and structure should accompany these changes to influence cell proliferation, delineation of tissues and acquisition of cell migration capabilities. Retinoic acid plays an important role in pre-patterning of the early embryo. Retinoic acid can be used in vitro to induce differentiation, causing pluripotent and multipotent cells to become increasingly lineage restricted. We examined retinoic acid-induced changes in the cellular proteoglycan composition of the well-characterized teratocarcinoma line NCCIT. Our analysis revealed changes in the abundance of transcripts for genes encoding core proteins, enzymes that are responsible for early and late linkage region biosynthesis, as well as enzymes for GAG chain extension and modification. Transcript levels for genes encoding core proteins used as backbones for polysaccharide synthesis revealed highly significant increases in expression of lumican and decorin, 1,500-fold and 2,800-fold, respectively. Similarly, glypican 3, glypican 5, versican and glypican 6 showed increases between 5 and 70-fold. Significant decreases in biglycan, serglycin, glypican 4, aggrecan, neurocan, CD74 and glypican 1 were observed. Disaccharide analysis of the glycans in heparin/heparan sulfate and chondroitin/dermatan sulfate revealed retinoic acid-induced changes restricted to chondroitin/dermatan sulfate glycans. Our study provides the first detailed analysis of changes in the glycosaminoglycan profile of human pluripotent cells upon treatment with the retinoic acid morphogen.

The interaction of kinesin-like proteins (Klps) with microtubules or α/β-tubulin heterodimers is fundamental to the mitotic mechanism through regulation of microtubule organization and dynamics. Since the discovery of conventional Kinesin in 1985, and kinesin-like proteins (Klps) beginning in 1990, information on the molecular nature of this interaction has been vague. In part its analysis has awaited elegant crystallographic data that now allows us to visualize, albeit still at low resolution, the site of interaction of monomeric and dimeric Klps on microtubules. In addition the availability of sequenced genomes provides bioinformatic profiling of conserved tubulin elements. This lab is investigating determinants of dimeric Klp/tubulin interactions. We recently defined for the first time, since the discovery of kinesin over 20 years ago, the binding site on tubulin for a Klp. That tubulin is the microtubule organizing center (MTOC) protein γ-tubulin and the Klp, dimeric Kinesin-14 Pkl1. Kinesin-14 Klps are ubiquitous in eukaryotes and oppose spindle bipolarity mediated by the Kinesin-5 family. The Kinesin-14/γ-tubulin interaction is effective as a prominent mechanism to oppose spindle bipolarity. We demonstrated that its disruption, by mutating the γ-tubulin site for Kinesin-14 binding, restored spindle bipolarity and viability in a normally impaired Kinesin-5 cut7-22ts strain. We are examining additional molecular features of the Kinesin-14/γ-tubulin binding site and determining its relationship to Klp/β-tubulin interactions. By site-directed mutagenesis we have altered similar residues in β-tubulin helix 11 and are generating chimeric β- and γ-tubulins with switched helix 11 domains. Analysis of the tubulin derivatives in vivo and in vitro will allow us to determine affects on Klp/tubulin interactions of both plus- and minus-end directed Klps and on microtubule dynamics and organization.

CPCI, the principal regulatory protein required for cross-pathway control of amino acid biosynthetic genes in Neurospora crassa, contains a domain similar to the DNA-binding domain of GCN4, the corresponding general regulator in Saccharomyces cerevisiae. We examined binding by CPC1 synthesized in vitro and by CPC1 present in N. crassa whole-cell extracts. CPCI from both sources was shown to bind to the DNA sequence 5'-ATGACTCAT-3', which is also the preferred recognition sequence of GCN4, CPC1 was confirmed as the source of DNA-binding activity in extracts by immunoblotting. Slightly mobility differences between DNA complexes containing CPCI synthesized in vitro and CPC1 in mycelial extracts were observed. Analyses of N. crassa extracts from different stages of asexual development revealed that CPC1 was abundant immediately following spore germination and through early mycelial growth but was scarce subsequently. CPC1 levels could be increased at any time by imposing amino acid starvation. Copies of the CPC1 response element are located upstream of several genes regulated by cross-pathway control, including cpc-1 itself.

Formation of a mitotic spindle occurs almost effortlessly as cells cycle and is essential for chromosome segregation. Early in a cell cycle, the replication of centrosomes or spindle pole bodies at G1/S leaves mother and daughter poles in close association until forced apart into a bipolar arrangement at mitotic onset. Kinesin-like proteins (Klps) generate a variety of forces that contribute to the timing, formation and maintenance of spindle bipolarity. The ability of two key players, Kinesin-5 and Kinesin-14, to cross-link both parallel and anti-parallel microtubules has led to emphasis on spindle microtubule interactions in the spindle assembly mechanism. Recent identification of a Kinesin-14 binding site on gamma-tubulin, a ubiquitous component of microtubule organizing centers (MTOCs) at poles combined with the ability of changes to MTOC complexes to regulate bipolarity, now shifts an inquisitive eye to pole mechanisms and to the complexities evolving around MTOCs.

Bipolar spindle assembly is essential to genomic stability in dividing cells. Centrosomes or spindle pole bodies duplicated earlier at G(1)/S remain adjacent until triggered at mitotic onset to become bipolar. Pole reorientation is stabilized by microtubule interdigitation but mechanistic details for bipolarity remain incomplete. To investigate the contribution of spindle pole microtubule organizing center (MTOC) proteins in bipolarity, we applied genetic, structural and molecular biochemical analysis along with timelapse microscopy. Spindle formation was followed by an in vivo growth assay with the conditional allele cut7-22(ts), encoding fission yeast mitotic Kinesin-5, essential for bipolarity. By analysis of double and triple mutant strains of MTOC alleles and cut7-22(ts) we found that stabilized microtubules or increased bundling can rescue cut7-22(ts) associated bipolarity defects. These changes to microtubule dynamics and organization occurred through two surface domains on gamma-tubulin, a helix 11 domain and an adjacent site for binding MTOC protein Alp4. We demonstrate that Kinesin-14 Pkl1, known to oppose bipolarity, can bind to gamma-tubulin at helix 11 and that mutation of either of two conserved residues in helix 11 can impair Kinesin-14 binding. Altering the Alp4/gamma-tubulin interaction, conserved residues in helix 11 or deletion of pkl1 each are sufficient to rescue bipolarity in our cut7-22(ts) strain. Our findings provide novel insights into regulation of the bipolar mechanism through the MTOC complex.

During evolution of fungi, the separate tryptophan synthetase alpha and beta polypeptides of bacteria appear to have been fused in the order alpha-beta rather than the beta-alpha order that would be predicted from the order of the corresponding structural genes in all bacteria. We have fused the tryptophan synthetase polypeptides of Escherichia coli in both orders, alpha-beta and beta-alpha, with and without a short connecting (con) sequence, to explore possible explanations for the domain arrangement in fungi. We find that proteins composed of any of the four fused polypeptides, beta-alpha, beta-con-alpha, alpha-beta, and alpha-con-beta, are highly active enzymatically. However, only the alpha-beta and alpha-con-beta proteins are as active as the wild type enzyme. All four fusion proteins appear to be less soluble in vivo than the wild type enzyme; this abnormal characteristic is minimal for the alpha-con-beta enzyme. The alpha and beta domains of the four fusion polypeptides were not appreciably more heat labile than the wild type polypeptides. Competition experiments with mutant tryptophan synthetase alpha protein, and the fusion proteins suggest that in each fusion protein the joined alpha and beta domains have a functional tunnel connecting their alpha and beta active sites. Three tryptophan synthetase beta'-alpha fusion proteins were examined in which the carboxyl-terminal segment of the wild type beta polypeptide was deleted and replaced by a shorter, unnatural sequence. The resulting deletion fusion proteins were enzymatically inactive and were found predominantly in the cell debris. Evaluation of our findings in relation to the three-dimensional structure of the tryptophan synthetase enzyme complex of Salmonella typhimurium (5) and the results of mutational analyses with E. coli suggest that tryptophan synthetase may have evolved via an alpha-beta rather than a beta-alpha fusion because in beta-alpha fusions the amino-terminal helix of the alpha chain cannot assume the conformation required for optimal enzymatic activity.

Signaling pathways within the mitotic mechanism temporally orchestrate spindle assembly with chromosome capture and alignment, and then coordinate initiation of chromosome segregation with spindle breakdown and cytokinesis for reproductive success. Kinetochore localized Mad2p acts in the spindle assembly checkpoint pathway during prophase and prometaphase to monitor bipolar attachment of chromosomes to spindle microtubules as well as proper tension at kinetochores. Once established, Mad2p is not degraded, but instead transits to spindle poles preceding the metaphase/anaphase transition in human and yeast cells. Whether conserved relocalization of Mad2p to poles is a final step in the spindle assembly checkpoint pathway or whether the post-metaphase transition allows Mad2p to cooperate in anaphase events leading to mitotic exit has been unknown. We examined post-metaphase localization of Mad2p in fission yeast. Our observations indicate an extended signaling pathway for Mad2p that includes kinetochore to bipolar localization at spindle poles, then additional transitions from bipolar to unipolar to equatorial. We determined that Mad2p associates with the microtubule organizing center complex through direct binding to Alp4p and that microtubule motor proteins Kinesin-14 Pkl1 and Dynein contribute to Mad2p anaphase transitions. At anaphase B onset, bipolar to unipolar transitions of both Mad2p and the septation inititiation network (SIN) kinase Cdc7 are observed. We determined that Mad2p and Cdc7p transitions monitor different events in anaphase, but that neither are required for anaphase B initiation. Our findings indicate that altered Mad2p anaphase spindle localizations can reflect changes in spindle function during mitotic exit that could contribute to fidelity in anaphase events.