Targeting Solid Cancers with a Cancer-Specific Monoclonal Antibody to Surface Expressed Aberrantly O-glycosylated Proteins.

The lack of antibodies with sufficient cancer selectivity is currently limiting the treatment of solid tumors by immunotherapies. Most current immunotherapeutic targets are tumor-associated antigens that are also found in healthy tissues and often do not display sufficient cancer selectivity to be used as targets for potent antibody-based immunotherapeutic treatments, such as chimeric antigen receptor (CAR) T cells. Many solid tumors, however, display aberrant glycosylation that results in expression of tumor-associated carbohydrate antigens that are distinct from healthy tissues. Targeting aberrantly glycosylated glycopeptide epitopes within existing or novel glycoprotein targets may provide the cancer selectivity needed for immunotherapy of solid tumors. However, to date only a few such glycopeptide epitopes have been targeted. Here, we used O-glycoproteomics data from multiple cell lines to identify a glycopeptide epitope in CD44v6, a cancer-associated CD44 isoform, and developed a cancer-specific mAb, 4C8, through a glycopeptide immunization strategy. 4C8 selectively binds to Tn-glycosylated CD44v6 in a site-specific manner with low nanomolar affinity. 4C8 was shown to be highly cancer specific by IHC of sections from multiple healthy and cancerous tissues. 4C8 CAR T cells demonstrated target-specific cytotoxicity in vitro and significant tumor regression and increased survival in vivo. Importantly, 4C8 CAR T cells were able to selectively kill target cells in a mixed organotypic skin cancer model having abundant CD44v6 expression without affecting healthy keratinocytes, indicating tolerability and safety.

Purification and Fluorescent Labeling of Tubulin from Xenopus laevis Egg Extracts.

For many years, microtubule research has depended on tubulin purified from cow and pig brains, which may not be ideal for experiments using proteins or extracts from non-brain tissues and cold-blooded organisms. Here, we describe a method to purify functional tubulin from the eggs of the frog, Xenopus laevis. This tubulin has many benefits for the study of microtubules and microtubule based structures assembled in vitro at room temperature. Frog tubulin lacks many of the highly stabilizing posttranslational modifications present in pig brain-derived tubulin, and polymerizes efficiently at room temperature. In addition, fluorescently labeled frog egg tubulin incorporates into meiotic spindles assembled in egg extract more efficiently than brain tubulin, and is thus superior as a probe for Xenopus egg extract experiments. Frog egg tubulin will provide excellent opportunities to identify active nucleation complexes and revisit microtubule polymerization dynamics in vitro.

The Nuclear Proteome of a Vertebrate.

The composition of the nucleoplasm determines the behavior of key processes such as transcription, yet there is still no reliable and quantitative resource of nuclear proteins. Furthermore, it is still unclear how the distinct nuclear and cytoplasmic compositions are maintained. To describe the nuclear proteome quantitatively, we isolated the large nuclei of frog oocytes via microdissection and measured the nucleocytoplasmic partitioning of ∼9,000 proteins by mass spectrometry. Most proteins localize entirely to either nucleus or cytoplasm; only ∼17% partition equally. A protein's native size in a complex, but not polypeptide molecular weight, is predictive of localization: partitioned proteins exhibit native sizes larger than ∼100 kDa, whereas natively smaller proteins are equidistributed. To evaluate the role of nuclear export in maintaining localization, we inhibited Exportin 1. This resulted in the expected re-localization of proteins toward the nucleus, but only 3% of the proteome was affected. Thus, complex assembly and passive retention, rather than continuous active transport, is the dominant mechanism for the maintenance of nuclear and cytoplasmic proteomes.

Spindle-to-cortex communication in cleaving, polyspermic Xenopus eggs.

Mitotic spindles specify cleavage planes in early embryos by communicating their position and orientation to the cell cortex using microtubule asters that grow out from the spindle poles during anaphase. Chromatin also plays a poorly understood role. Polyspermic fertilization provides a natural experiment in which aster pairs from the same spindle (sister asters) have chromatin between them, whereas asters pairs from different spindles (nonsisters) do not. In frogs, only sister aster pairs induce furrows. We found that only sister asters recruited two conserved furrow-inducing signaling complexes, chromosome passenger complex (CPC) and Centralspindlin, to a plane between them. This explains why only sister pairs induce furrows. We then investigated factors that influenced CPC recruitment to microtubule bundles in intact eggs and a cytokinesis extract system. We found that microtubule stabilization, optimal starting distance between asters, and proximity to chromatin all favored CPC recruitment. We propose a model in which proximity to chromatin biases initial CPC recruitment to microtubule bundles between asters from the same spindle. Next a positive feedback between CPC recruitment and microtubule stabilization promotes lateral growth of a plane of CPC-positive microtubule bundles out to the cortex to position the furrow.

Using supported bilayers to study the spatiotemporal organization of membrane-bound proteins.

Cell division in prokaryotes and eukaryotes is commonly initiated by the well-controlled binding of proteins to the cytoplasmic side of the cell membrane. However, a precise characterization of the spatiotemporal dynamics of membrane-bound proteins is often difficult to achieve in vivo. Here, we present protocols for the use of supported lipid bilayers to rebuild the cytokinetic machineries of cells with greatly different dimensions: the bacterium Escherichia coli and eggs of the vertebrate Xenopus laevis. Combined with total internal reflection fluorescence microscopy, these experimental setups allow for precise quantitative analyses of membrane-bound proteins. The protocols described to obtain glass-supported membranes from bacterial and vertebrate lipids can be used as starting points for other reconstitution experiments. We believe that similar biochemical assays will be instrumental to study the biochemistry and biophysics underlying a variety of complex cellular tasks, such as signaling, vesicle trafficking, and cell motility.

Microtubule nucleation remote from centrosomes may explain how asters span large cells.

A major challenge in cell biology is to understand how nanometer-sized molecules can organize micrometer-sized cells in space and time. One solution in many animal cells is a radial array of microtubules called an aster, which is nucleated by a central organizing center and spans the entire cytoplasm. Frog (here Xenopus laevis) embryos are more than 1 mm in diameter and divide with a defined geometry every 30 min. Like smaller cells, they are organized by asters, which grow, interact, and move to precisely position the cleavage planes. It has been unclear whether asters grow to fill the enormous egg by the same mechanism used in smaller somatic cells, or whether special mechanisms are required. We addressed this question by imaging growing asters in a cell-free system derived from eggs, where asters grew to hundreds of microns in diameter. By tracking marks on the lattice, we found that microtubules could slide outward, but this was not essential for rapid aster growth. Polymer treadmilling did not occur. By measuring the number and positions of microtubule ends over time, we found that most microtubules were nucleated away from the centrosome and that interphase egg cytoplasm supported spontaneous nucleation after a time lag. We propose that aster growth is initiated by centrosomes but that asters grow by propagating a wave of microtubule nucleation stimulated by the presence of preexisting microtubules.

Spatial organization of cytokinesis signaling reconstituted in a cell-free system.

During animal cell division, the cleavage furrow is positioned by microtubules that signal to the actin cortex at the cell midplane. We developed a cell-free system to recapitulate cytokinesis signaling using cytoplasmic extract from Xenopus eggs. Microtubules grew out as asters from artificial centrosomes and met to organize antiparallel overlap zones. These zones blocked the interpenetration of neighboring asters and recruited cytokinesis midzone proteins, including the chromosomal passenger complex (CPC) and centralspindlin. The CPC was transported to overlap zones, which required two motor proteins, Kif4A and a Kif20A paralog. Using supported lipid bilayers to mimic the plasma membrane, we observed the recruitment of cleavage furrow markers, including an active RhoA reporter, at microtubule overlaps. This system opens further approaches to understanding the biophysics of cytokinesis signaling.

Organization of early frog embryos by chemical waves emanating from centrosomes.

The large cells in early vertebrate development face an extreme physical challenge in organizing their cytoplasm. For example, amphibian embryos have to divide cytoplasm that spans hundreds of micrometres every 30 min according to a precise geometry, a remarkable accomplishment given the extreme difference between molecular and cellular scales in this system. How do the biochemical reactions occurring at the molecular scale lead to this emergent behaviour of the cell as a whole? Based on recent findings, we propose that the centrosome plays a crucial role by initiating two autocatalytic reactions that travel across the large cytoplasm as chemical waves. Waves of mitotic entry and exit propagate out from centrosomes using the Cdk1 oscillator to coordinate the timing of cell division. Waves of microtubule-stimulated microtubule nucleation propagate out to assemble large asters that position spindles for the following mitosis and establish cleavage plane geometry. By initiating these chemical waves, the centrosome rapidly organizes the large cytoplasm during the short embryonic cell cycle, which would be impossible using more conventional mechanisms such as diffusion or nucleation by structural templating. Large embryo cells provide valuable insights to how cells control chemical waves, which may be a general principle for cytoplasmic organization.

Xenopus egg cytoplasm with intact actin.

We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

Glycogen-supplemented mitotic cytosol for analyzing Xenopus egg microtubule organization.

Undiluted cytoplasmic extract prepared from unfertilized Xenopus laevis eggs by low-speed centrifugation (CSF extracts) is useful for reconstitution of egg microtubule dynamics and meiosis-II spindle organization, but it suffers limitations for biochemical analysis due to abundant particulates. Here, we describe preparation and the use of fully clarified, undiluted mitotic cytosol derived from CSF extract. Addition of glycogen improves the ability of this cytosol to reconstitute microtubule organization, in part through improved energy metabolism. Using fully clarified, glycogen-supplemented mitotic cytosol, we reconstituted (i) stimulation of microtubule polymerization by Ran.GTP (Groen, Coughlin, & Mitchison, 2011; Ohba, Nakamura, Nishitani, & Nishimoto, 1999) and (ii) self-organization of highly regular bipolar arrays of taxol-stabilized microtubules that we termed "pineapples" (Mitchison, Nguyen, Coughlin, & Groen, 2013). Both systems will be useful for biochemical dissection of spindle assembly mechanisms. We also describe reliable small-scale methods for preparing fluorescent antibody probes that can be used for live imaging in egg extract systems as well as standard immunofluorescence.

Electron microscopy of microtubule cytoskeleton assembly in vitro.

Cell-free cytoplasm isolated from meiotic Xenopus egg extracts reconstitutes microtubule phenomena in vitro. These crude extracts assemble bipolar meiotic spindles and are readily fractionated for biochemical assays, providing a good tool to dissect molecular mechanism. We developed techniques for immunoelectron microscopy of microtubule structures assembled in perfusion chambers and in solution.

Self-organization of stabilized microtubules by both spindle and midzone mechanisms in Xenopus egg cytosol.

Previous study of self-organization of Taxol-stabilized microtubules into asters in Xenopus meiotic extracts revealed motor-dependent organizational mechanisms in the spindle. We revisit this approach using clarified cytosol with glycogen added back to supply energy and reducing equivalents. We added probes for NUMA and Aurora B to reveal microtubule polarity. Taxol and dimethyl sulfoxide promote rapid polymerization of microtubules that slowly self-organize into assemblies with a characteristic morphology consisting of paired lines or open circles of parallel bundles. Minus ends align in NUMA-containing foci on the outside, and plus ends in Aurora B-containing foci on the inside. Assemblies have a well-defined width that depends on initial assembly conditions, but microtubules within them have a broad length distribution. Electron microscopy shows that plus-end foci are coated with electron-dense material and resemble similar foci in monopolar midzones in cells. Functional tests show that two key spindle assembly factors, dynein and kinesin-5, act during assembly as they do in spindles, whereas two key midzone assembly factors, Aurora B and Kif4, act as they do in midzones. These data reveal the richness of self-organizing mechanisms that operate on microtubules after they polymerize in meiotic cytoplasm and provide a biochemically tractable system for investigating plus-end organization in midzones.

Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2.

The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence has suggested that microtubules also might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires γ-tubulin and augmin and is stimulated by factors previously implicated in chromatin-stimulated nucleation, guanosine triphosphate(GTP)-bound Ran and its effector, TPX2. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance.

Microtubule assembly in meiotic extract requires glycogen.

The assembly of microtubules during mitosis requires many identified components, such as γ-tubulin ring complex (γ-TuRC), components of the Ran pathway (e.g., TPX2, HuRP, and Rae1), and XMAP215/chTOG. However, it is far from clear how these factors function together or whether more factors exist. In this study, we used biochemistry to attempt to identify active microtubule nucleation protein complexes from Xenopus meiotic egg extracts. Unexpectedly, we found both microtubule assembly and bipolar spindle assembly required glycogen, which acted both as a crowding agent and as metabolic source glucose. By also reconstituting microtubule assembly in clarified extracts, we showed microtubule assembly does not require ribosomes, mitochondria, or membranes. Our clarified extracts will provide a powerful tool for activity-based biochemical fractionations for microtubule assembly.

Spindle assembly in the absence of a RanGTP gradient requires localized CPC activity.

During animal cell division, a gradient of GTP-bound Ran is generated around mitotic chromatin. It is generally accepted that this RanGTP gradient is essential for organizing the spindle, because it locally activates critical spindle assembly factors. Here, we show in Xenopus laevis egg extract, where the gradient is best characterized, that spindles can assemble in the absence of a RanGTP gradient. Gradient-free spindle assembly occurred around sperm nuclei but not around chromatin-coated beads and required the chromosomal passenger complex (CPC). Artificial enrichment of CPC activity within hybrid bead arrays containing both immobilized chromatin and the CPC supported local microtubule assembly even in the absence of a RanGTP gradient. We conclude that RanGTP and the CPC constitute the two major molecular signals that spatially promote microtubule polymerization around chromatin. Furthermore, we hypothesize that the two signals mainly originate from discreet physical sites on the chromosomes to localize microtubule assembly around chromatin: a RanGTP signal from any chromatin and a CPC-dependent signal predominantly generated from centromeric chromatin.

Functional overlap of microtubule assembly factors in chromatin-promoted spindle assembly.

Distinct pathways from centrosomes and chromatin are thought to contribute in parallel to microtubule nucleation and stabilization during animal cell mitotic spindle assembly, but their full mechanisms are not known. We investigated the function of three proposed nucleation/stabilization factors, TPX2, gamma-tubulin and XMAP215, in chromatin-promoted assembly of anastral spindles in Xenopus laevis egg extract. In addition to conventional depletion-add back experiments, we tested whether factors could substitute for each other, indicative of functional redundancy. All three factors were required for microtubule polymerization and bipolar spindle assembly around chromatin beads. Depletion of TPX2 was partially rescued by the addition of excess XMAP215 or EB1, or inhibiting MCAK (a Kinesin-13). Depletion of either gamma-tubulin or XMAP215 was partially rescued by adding back XMAP215, but not by adding any of the other factors. These data reveal functional redundancy between specific assembly factors in the chromatin pathway, suggesting individual proteins or pathways commonly viewed to be essential may not have entirely unique functions.

How does a millimeter-sized cell find its center?

Microtubules play a central role in centering the nucleus or mitotic spindle in eukaryotic cells. However, despite common use of microtubules for centering, physical mechanisms can vary greatly, and depend on cell size and cell type. In the small fission yeast cells, the nucleus can be centered by pushing forces that are generated when growing microtubules hit the cell boundary. This mechanism may not be possible in larger cells, because the compressive force that microtubules can sustain are limited by buckling, so maximal force decreases with microtubule length. In a well-studied intermediate sized cell, the C. elegans fertilized egg, centrosomes are centered by cortex-attached motors that pull on microtubules. This mechanism is widely assumed to be general for larger cells. However, re-evaluation of classic experiments in a very large cell, the fertilized amphibian egg, argues against such generality. In these large eggs, movement of asters away from a part of the cell boundary that they are touching cannot be mediated by cortical pulling, because the astral microtubules are too short to reach the opposite cell boundary. Additionally, Herlant and Brachet discovered a century ago that multiple asters within a single egg center relative to the cell boundary, but also relative to each other. Here, we summarize current understanding of microtubule organization during the first cell cycle in a fertilized Xenopus egg, discuss how microtubule asters move towards the center of this very large cell, and how multiple asters shape and position themselves relative to each other.

Spindle fusion requires dynein-mediated sliding of oppositely oriented microtubules.

BACKGROUND: Bipolar spindle assembly is critical for achieving accurate segregation of chromosomes. In the absence of centrosomes, meiotic spindles achieve bipolarity by a combination of chromosome-initiated microtubule nucleation and stabilization and motor-driven organization of microtubules. Once assembled, the spindle structure is maintained on a relatively long time scale despite the high turnover of the microtubules that comprise it. To study the underlying mechanisms responsible for spindle assembly and steady-state maintenance, we used microneedle manipulation of preassembled spindles in Xenopus egg extracts. RESULTS: When two meiotic spindles were brought close enough together, they interacted, creating an interconnected microtubule structure with supernumerary poles. Without exception, the perturbed system eventually re-established bipolarity, forming a single spindle of normal shape and size. Bipolar spindle fusion was blocked when cytoplasmic dynein function was perturbed, suggesting a critical role for the motor in this process. The fusion of Eg5-inhibited monopoles also required dynein function but only occurred if the initial interpolar separation was less than twice the microtubule radius of a typical monopole. CONCLUSIONS: Our experiments uniquely illustrate the architectural plasticity of the spindle and reveal a robust ability of the system to attain a bipolar morphology. We hypothesize that a major mechanism driving spindle fusion is dynein-mediated sliding of oppositely oriented microtubules, a novel function for the motor, and posit that this same mechanism might also be involved in normal spindle assembly and homeostasis.

Evidence for an upper limit to mitotic spindle length.

Size specification of macromolecular assemblies in the cytoplasm is poorly understood [1]. In principle, assemblies could scale with cell size or use intrinsic mechanisms. For the mitotic spindle, scaling with cell size is expected, because the function of this assembly is to physically move sister chromatids into the center of nascent daughter cells. Eggs of Xenopus laevis are among the largest cells known that cleave completely during cell division. Cell length in this organism changes by two orders of magnitude ( approximately 1200 microm to approximately 12 microm) while it develops from a fertilized egg into a tadpole [2]. We wondered whether, and how, mitotic spindle length and morphology adapt to function at these different length scales. Here, we show that spindle length increases with cell length in small cells, but in very large cells spindle length approaches an upper limit of approximately 60 microm. Further evidence for an upper limit to spindle length comes from an embryonic extract system that recapitulates mitotic spindle assembly in a test tube. We conclude that early mitotic spindle length in Xenopus laevis is uncoupled from cell length, reaching an upper bound determined by mechanisms that are intrinsic to the spindle.

A novel small-molecule inhibitor reveals a possible role of kinesin-5 in anastral spindle-pole assembly.

The tetrameric plus-end-directed motor, kinesin-5, is essential for bipolar spindle assembly. Small-molecule inhibitors of kinesin-5 have been important tools for investigating its function, and some are currently under evaluation as anti-cancer drugs. Most inhibitors reported to date are ;non-competitive' and bind to a specific site on the motor head, trapping the motor in an ADP-bound state in which it has a weak but non-zero affinity for microtubules. Here, we used a novel ATP-competitive inhibitor, FCPT, developed at Merck (USA). We found that it induced tight binding of kinesin-5 onto microtubules in vitro. Using Xenopus egg-extract spindles, we found that FCPT not only blocked poleward microtubule sliding but also selectively induced loss of microtubules at the poles of bipolar spindles (and not asters or monoasters). We also found that the spindle-pole proteins TPX2 and gamma-tubulin became redistributed to the spindle equator, suggesting that proper kinesin-5 function is required for pole assembly.