The histopathology of bronchial asthma is associated with structural changes within the airways, including subepithelial fibrosis, as well as chronic eosinophilic inflammation. The mechanisms responsible for this tissue remodeling, and in particular the role of inflammatory cells, remain to be established. Transforming growth factor-beta (TGF-beta) is a potent profibrotic cytokine which may contribute to the thickening of the reticular lamina by the deposition of collagen fibers. To investigate the molecular mechanisms underlying these structural changes, we have investigated the expression of TGF-beta1 mRNA and immunoreactivity within the bronchial mucosa of mild to severe asthmatic individuals and normal control subjects using the techniques of in situ hybridization and immunocytochemistry. As eosinophils are prominent within the asthmatic airway and are known to synthesize pro-inflammatory cytokines, the presence of TGF-beta1 mRNA and immunoreactive protein in eosinophils was also examined. Asthmatic individuals exhibited a greater expression of TGF-beta1 mRNA and immunoreactivity in the airways submucosa than normal control subjects (P < 0.05), and these increases were directly related to the severity of the disorder. The extent of airways fibrosis, as detected histochemically, was also increased in asthmatics compared with normal control subjects (P < 0.005). In asthmatic subjects, the presence of subepithelial fibrosis was associated with the severity of the disease and correlated with the decline in forced expiratory volume in 1 s (r2 = 0.78; P < 0.05). Within the asthmatic airways, EG2-positive eosinophils represented the major source of TGF-beta1 mRNA and immunoreactivity. These results provide evidence that TGF-beta1 may play a role in the fibrotic changes occurring within asthmatic airways and that activated eosinophils are a major source of this cytokine.

The stable propagation of genetic material during cell division depends on the congression of chromosomes to the spindle equator before the cell initiates anaphase. It is generally assumed that congression requires that chromosomes are connected to the opposite poles of the bipolar spindle ("bioriented"). In mammalian cells, we found that chromosomes can congress before becoming bioriented. By combining the use of reversible chemical inhibitors, live-cell light microscopy, and correlative electron microscopy, we found that monooriented chromosomes could glide toward the spindle equator alongside kinetochore fibers attached to other already bioriented chromosomes. This congression mechanism depended on the kinetochore-associated, plus end-directed microtubule motor CENP-E (kinesin-7).Successful cell division requires proper "biorientation" of chromosomes, whereby microtubule bundles (K fibers) connect sister kinetochores of each chromosome to opposite spindle poles (1). Biorientation errors are linked to chromosome loss and cancers (2). Formation of sister K fibers occurs asynchronously (3), and once a kinetochore captures microtubules growing from a spindle pole, the chromosome is transported toward this pole and becomes "monooriented" (4). Monooriented chromosomes remain near the spindle pole for variable times (3, 4) until they suddenly "congress" to the spindle equator. Current models of mitotic spindle formation (5, 6) postulate that chromosome congression occurs as the result of biorientation (7).We followed movements of individual chromosomes in mammalian cells by differential interference contrast (DIC) time-lapse microscopy (8 Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript oscillations that occur toward and away from spindle poles, we frequently observed monooriented chromosomes making direct movements to the metaphase plate as if they were attempting to congress ( fig. S1). Centromeres on these congressing chromosomes were frequently stretched, which indicated force generation by the leading kinetochore (Movie S1). However, these movements did not always result in a stable alignment on the metaphase plate, because chromosomes often returned to the spindle pole after a 3-to 4-μm excursion. This chromosome behavior was observed in essentially every cell we imaged and has also been previously reported (9-12). To determine whether these chromosomes were bioriented, we followed mitotic cells by DIC microscopy until one of the chromosomes exhibited an extended linear movement toward the metaphase plate, and we fixed the cell when the chromosome had almost reached the metaphase plate (~5 to 7 μm from the proximal spindle pole) (Fig.

systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens. Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts..

Actin polymerization is required for the generation of motile force at the leading edge of both lamellipodia and filopodia and also at the surface of motile intracellular bacterial pathogens such as Listeria monocytogenes. Local catalysis of actin filament polymerization is accomplished in L. monocytogenes by the bacterial protein ActA. Polystyrene beads coated with purified ActA protein can undergo directional movement in an actin-rich cytoplasmic extract. Thus, the actin polymerization-based motility generated by ActA can be used to move nonbiological cargo, as has been demonstrated for classical motor molecules such as kinesin and myosin. Initiation of unidirectional movement of a symmetrically coated particle is a function of bead size and surface protein density. Small beads (<0.5 m in diameter) initiate actin-based motility when local asymmetries are built up by random f luctuations of actin filament density or by thermal motion, demonstrating the inherent ability of the dynamic actin cytoskeleton to spontaneously self-organize into a polar structure capable of generating unidirectional force. Larger beads (up to 2 m in diameter) can initiate movement only if surface asymmetry is introduced by coating the beads on one hemisphere. This explains why the relatively large L. monocytogenes requires polar distribution of ActA on its surface to move.Listeria monocytogenes is a Gram-positive intracellular bacterial pathogen that moves rapidly through host cells or cytoplasmic extracts, propelled by actin hijacked from the host cell (1-3). Moving bacteria are associated with an actin-rich structure that resembles the tail of a comet (4). The actin filaments within the comet tail remain fixed in space as the bacterium moves, and the rate of bacterial movement is tightly correlated with the rate of actin filament polymerization at the front of the comet tail (5, 6). Because no motor proteins have been found to be involved in this form of motility, filament elongation is thought to provide the force necessary to propel the bacterium through the cytoplasm (7). The comet tail structure is functionally similar to the actin meshwork in the lamellipodia of a locomoting eukaryotic cell, and the surface of the bacterium is analogous to the leading edge. Therefore, insights into the mechanism of actin-based bacterial movement should help explain the mechanisms of eukaryotic actin-based motility.A bacterial comet tail is made up of short (0.2-0.3 m) actin filaments crosslinked together to form a dense cylindrical structure (4). Other intracytoplasmic pathogens form similar comet tail structures and undergo actin-based motility, including the unrelated bacteria Shigella flexneri (8) and Rickettsia spp. (9) and vaccina virus (10). In uninfected cells and cytoplasmic extracts, actin-rich comet tails are associated with moving endosomes (J. Heuser, personal communication) and phospholipid vesicles (11). Although much is understood about the biochemical mechanisms of catalyzing actin filament polymerization in several of th...

Merotelic kinetochore orientation is a kinetochore misattachment in which a single kinetochore is attached to microtubules from both spindle poles instead of just one. It can be favored in specific circumstances, is not detected by the mitotic checkpoint, and induces lagging chromosomes in anaphase. In mammalian cells, it occurs at high frequency in early mitosis, but few anaphase cells show lagging chromosomes. We developed live-cell imaging methods to determine whether and how the mitotic spindle prevents merotelic kinetochores from producing lagging chromosomes. We found that merotelic kinetochores entering anaphase never lost attachment to the spindle poles; they remained attached to both microtubule bundles, but this did not prevent them from segregating correctly. The two microtubule bundles usually showed different fluorescence intensities, the brighter bundle connecting the merotelic kinetochore to the correct pole. During anaphase, the dimmer bundle lengthened much more than the brighter bundle as spindle elongation occurred. This resulted in correct segregation of the merotelically oriented chromosome. We propose a model based on the ratios of microtubules to the correct versus incorrect pole for how anaphase spindle dynamics and microtubule polymerization at kinetochores prevent potential segregation errors deriving from merotelic kinetochore orientation.

Proper positioning of the cell division plane during mitosis is essential for determining the size and position of the two daughter cells--a critical step during development and cell differentiation. A bipolar microtubule array has been proposed to be a minimum requirement for furrow positioning in mammalian cells, with furrows forming at the site of microtubule plus-end overlap between the spindle poles. Observations in other species have suggested, however, that this may not be true. Here we show, by inducing mammalian tissue cells with monopolar spindles to enter anaphase, that furrow formation in cultured mammalian cells does not require a bipolar spindle. Unexpectedly, cytokinesis occurs at high frequency in monopolar cells. Division always occurs at a cortical position distal to the chromosomes. Analysis of microtubules during cytokinesis in cells with monopolar and bipolar spindles shows that a subpopulation of stable microtubules extends past chromosomes and binds to the cell cortex at the site of furrow formation. Our data are consistent with a model in which chromosomes supply microtubules with factors that promote microtubule stability and furrowing.

When chromosomes are aligned and bioriented at metaphase, the elastic stretch of centromeric chromatin opposes pulling forces exerted on sister kinetochores by the mitotic spindle. Here we show that condensin ATPase activity is an important regulator of centromere stiffness and function. Condensin depletion decreases the stiffness of centromeric chromatin by 50% when pulling forces are applied to kinetochores. However, condensin is dispensable for the normal level of compaction (rest length) of centromeres, which probably depends on other factors that control higher-order chromatin folding. Kinetochores also do not require condensin for their structure or motility. Loss of stiffness caused by condensindepletion produces abnormal uncoordinated sister kinetochore movements, leads to an increase in Mad2(؉) kinetochores near the metaphase plate and delays anaphase onset. INTRODUCTIONCentromeric chromatin is a special region of chromosomes that has important mechanical and signaling functions in mitosis (Pidoux and Allshire, 2005;Ekwall, 2007;Cheeseman and Desai, 2008;Vagnarelli et al., 2008). In metaphase, pulling forces generated by interactions between spindle microtubules (MTs) and kinetochores are opposed by tension produced by centromeric chromatin stretch. Centromere and kinetochore tension and stretch are important for maintaining chromosome alignment (McIntosh et al., 2002), stabilizing kinetochore microtubule (kMT) attachments (Nicklas and Koch, 1969), spindle checkpoint signaling (Musacchio and Salmon, 2007;McEwen and Dong, 2009), and also for the back-to-back orientation of sister kinetochores (Loncarek et al., 2007). At least three independent factors have roles in the establishment of centromeric tension in metaphase: sister chromatid cohesion (Yeh et al., 2008), the elastic properties of chromatin (Houchmandzadeh et al., 1997;Almagro et al., 2004;Marko, 2008), and the higher order structure of the centromeric chromatin.Condensin is important for the architecture of mitotic chromosome arms (Coelho et al., 2003;Hudson et al., 2003;Hirota et al., 2004;Hirano, 2006), but it also localizes to centromeres (Saitoh et al., 1994;Gerlich et al., 2006), where condensin I, but not condensin II was reported to have a role in stabilizing the structure (Gerlich et al., 2006). It has recently been suggested that condensin could have a role in regulating the elastic behavior of centromeric chromatin. One study found that condensin I-depleted Drosophila chromosomes were unable to align at a metaphase plate, had distorted kinetochore structures, and lost elasticity of their centromeric chromatin (Oliveira et al., 2005). However a similar study in human cells reported that although loss of condensin I caused kinetochores to undergo abnormal movements, these movements were bidirectional (e.g., reversible; Gerlich et al., 2006).Even after the publication of those results, the regulation and functional significance of centromere stretch remained unknown. An elegant study in budding yeast went on to find that chromatin struct...

Polymerization of actin filaments is necessary for both protrusion of the leading edge of crawling cells and propulsion of certain intracellular pathogens, and it is sufficient for generating force for bacterial motility in vitro. Motile intracellular pathogens are associated with actin-rich comet tails containing many of the same molecular components present in lamellipodia, and this suggests that these two systems use a similar mechanism for motility. However, available structural evidence suggests that the organization of comet tails differs from that of lamellipodia. Actin filaments in lamellipodia form branched arrays, which are thought to arise by dendritic nucleation mediated by the Arp2/3 complex. In contrast, comet tails have been variously described as consisting of short, randomly oriented filaments, with a higher degree of alignment at the periphery, or as containing long, straight axial filaments with a small number of oblique filaments. Because the assembly of pathogen-associated comet tails has been used as a model system for lamellipodial protrusion, it is important to resolve this apparent discrepancy. Here, using a platinum replica approach, we show that actin filament arrays in comet tails in fact have a dendritic organization with the Arp2/3 complex localizing to Y-junctions as in lamellipodia. Thus, comet tails and lamellipodia appear to share a common dendritic nucleation mechanism for protrusive motility. However, comet tails differ from lamellipodia in that their actin filaments are usually twisted and appear to be under significant torsional stress.