Septin 7 interacts with centromere-associated protein E and is required for its kinetochore localization

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore. Septin (SEPT) belongs to a conserved family of polymerizing GTPases localized to the metaphase spindle during mitosis. Previous study showed that SEPT2 depletion results in chromosome mis-segregation correlated with a loss of centromere-associated protein E (CENP-E) from the kinetochores of congressing chromosomes (1). However, it has remained elusive as to whether CENP-E physically interacts with SEPT and how this interaction orchestrates chromosome segregation in mitosis. Here we show that SEPT7 is required for a stable kinetochore localization of CENP-E in HeLa and MDCK cells. SEPT7 stabilizes the kinetochore association of CENP-E by directly interacting with its C-terminal domain. The region of SEPT7 binding to CENP-E was mapped to its C-terminal domain by glutathione S-transferase pull-down and yeast two-hybrid assays. Immunofluorescence study shows that SEPT7 filaments distribute along the mitotic spindle and terminate at the kinetochore marked by CENP-E. Remarkably, suppression of synthesis of SEPT7 by small interfering RNA abrogated the localization of CENP-E to the kinetochore and caused aberrant chromosome segregation. These mitotic defects and kinetochore localization of CENP-E can be successfully rescued by introducing exogenous GFP-SEPT7 into the SEPT7-depleted cells. These SEPT7-suppressed cells display reduced tension at kinetochores of bi-orientated chromosomes and activated mitotic spindle checkpoint marked by Mad2 and BubR1 labelings on these misaligned chromosomes. These findings reveal a key role for the SEPT7-CENP-E interaction in the distribution of CENP-E to the kinetochore and achieving chromosome alignment. We propose that SEPT7 forms a link between kinetochore distribution of CENP-E and the mitotic spindle checkpoint.     

The Microtubule-dependent Motor Centromere–associated Protein E (CENP-E) Is an Integral Component of Kinetochore Corona Fibers That Link Centromeres to Spindle Microtubules

Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Using immunoelectron microscopy, CENP-E is shown to be an integral component of the kinetochore corona fibers that tether centromeres to the spindle. Immediately upon nuclear envelope fragmentation, an associated plus end motor trafficks cytoplasmic CENP-E toward chromosomes along astral microtubules that enter the nuclear volume. Before or concurrently with initial lateral attachment of spindle microtubules, CENP-E targets to the outermost region of the developing kinetochores. After stable attachment, throughout chromosome congression, at metaphase, and throughout anaphase A, CENP-E is a constituent of the corona fibers, extending at least 50 nm away from the kinetochore outer plate and intertwining with spindle microtubules. In congressing chromosomes, CENP-E is preferentially associated with (or accessible at) the stretched, leading kinetochore known to provide the primary power for chromosome movement. Taken together, this evidence strongly supports a model in which CENP-E functions in congression to tether kinetochores to the disassembling microtubule plus ends.     

Human NUF2 interacts with centromere-associated protein E and is essential for a stable spindle microtubule-kinetochore attachment

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here, we show that Homo sapiens (Hs) NUF2 is required for stable kinetochore localization of centromere-associated protein E (CENP-E) in HeLa cells. HsNUF2 specifies the kinetochore association of CENP-E by interacting with its C-terminal domain. The region of HsNUF2 binding to CENP-E was mapped to its C-terminal domain by glutathione S-transferase pulldown and yeast two-hybrid assays. Suppression of synthesis of HsNUF2 by small interfering RNA abrogated the localization of CENP-E to the kinetochore, demonstrating the requirement of HsNUF2 for CENP-E kinetochore localization. In addition, depletion of HsNUF2 caused aberrant chromosome segregation. These HsNUF2-suppressed cells displayed reduced tension at kinetochores of bi-orientated chromosomes. Double knockdown of CENP-E and HsNUF2 further abolished the tension at the kinetochores. Our results indicate that HsNUF2 and CENP-E are required for organization of stable microtubule-kinetochore attachment that is essential for faithful chromosome segregation in mitosis.     

CAMP (C13orf8, ZNF828) is a novel regulator of kinetochore-microtubule attachment

Proper attachment of microtubules to kinetochores is essential for accurate chromosome segregation. Here, we report a novel protein involved in kinetochore-microtubule attachment, chromosome alignment-maintaining phosphoprotein (CAMP) (C13orf8, ZNF828). CAMP is a zinc-finger protein containing three characteristic repeat motifs termed the WK, SPE, and FPE motifs. CAMP localizes to chromosomes and the spindle including kinetochores, and undergoes CDK1-dependent phosphorylation at multiple sites during mitosis. CAMP-depleted cells showed severe chromosome misalignment, which was associated with the poor resistance of K-fibres to the tension exerted upon establishment of sister kinetochore bi-orientation. We found that the FPE region, which is responsible for spindle and kinetochore localization, is essential for proper chromosome alignment. The C-terminal region containing the zinc-finger domains negatively regulates chromosome alignment, and phosphorylation in the FPE region counteracts this regulation. Kinetochore localization of CENP-E and CENP-F was affected by CAMP depletion, and by expressing CAMP mutants that cannot functionally rescue CAMP depletion, placing CENP-E and CENP-F as downstream effectors of CAMP. These data suggest that CAMP is required for maintaining kinetochore-microtubule attachment during bi-orientation.     

CENP-E--dependent BubR1 autophosphorylation enhances chromosome alignment and the mitotic checkpoint

How the state of spindle microtubule capture at the kinetochore is translated into mitotic checkpoint signaling remains largely unknown. In this paper, we demonstrate that the kinetochore-associated mitotic kinase BubR1 phosphorylates itself in human cells and that this autophosphorylation is dependent on its binding partner, the kinetochore motor CENP-E. This CENP-E-dependent BubR1 autophosphorylation at unattached kinetochores is important for a full-strength mitotic checkpoint to prevent single chromosome loss. Replacing endogenous BubR1 with a nonphosphorylatable BubR1 mutant, as well as depletion of CENP-E, the BubR1 kinase activator, results in metaphase chromosome misalignment and a decrease of Aurora B-mediated Ndc80 phosphorylation at kinetochores. Furthermore, expressing a phosphomimetic BubR1 mutant substantially reduces the incidence of polar chromosomes in CENP-E-depleted cells. Thus, the state of CENP-E-dependent BubR1 autophosphorylation in response to spindle microtubule capture by CENP-E is important for kinetochore function in achieving accurate chromosome segregation.     

TRAMM/TrappC12 plays a role in chromosome congression,kinetochore stability,and CENP-E recruitment

Chromosome congression requires the stable attachment of microtubules to chromosomes mediated by the kinetochore, a large proteinaceous structure whose mechanism of assembly is unknown. In this paper, we present the finding that a protein called TRAMM (formerly known as TrappC12) plays a role in mitosis. Depletion of TRAMM resulted in noncongressed chromosomes and arrested cells in mitosis. Small amounts of TRAMM associated with chromosomes, and its depletion affected the localization of some kinetochore proteins, the strongest effect being seen for CENP-E. TRAMM interacts with CENP-E, and depletion of TRAMM prevented the recruitment of CENP-E to the kinetochore. TRAMM is phosphorylated early in mitosis and dephosphorylated at the onset of anaphase. Interestingly, this phosphorylation/dephosphorylation cycle correlates with its association/disassociation with CENP-E. Finally, we demonstrate that a phosphomimetic form of TRAMM recruited CENP-E to kinetochores more efficiently than did the nonphosphorylatable mutant. Our study identifies a moonlighting function for TRAMM during mitosis and adds a new component that regulates kinetochore stability and CENP-E recruitment.     

hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells

Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore-microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.     

Neural Wiskott-Aldrich syndrome protein is required for accurate chromosome congression and segregation

The accurate distribution and segregation of replicated chromosomes through mitosis is crucial for cellular viability and development of organisms. Kinetochores are responsible for the proper congression and segregation of chromosomes. Here, we show that neural Wiskott-Aldrich syndrome protein (N-WASP) localizes to and forms a complex with kinetochores in mitotic cells. Depletion of NWASP by RNA interference causes chromosome misalignment, prolonged mitosis, and abnormal chromosomal segregation, which is associated with decreased proliferation of N-WASP-deficient cells. N-WASP-deficient cells display defects in the kinetochores recruitment of inner and outer kinetochore components, CENP-A, CENP-E, and Mad2. Live-cell imaging analysis of GFP-α-tubulin revealed that depletion of N-WASP impairs microtubule attachment to chromosomes in mitotic cells. All these results indicate that N-WASP plays a role in efficient assembly of kinetochores and attachment of microtubules to chromosomes, which is essential for accurate chromosome congression and segregation.     

Mitosin／CENP-F is a conserved kinetochore protein subjected to cyto-plasmic dynein-mediated poleward transport

Mitosin/CENP-F is a human nuclear protein transiently associated with the outer kinetochore plate in M phase and is involved in M phase progression. LEK1 and CMF1, which are its murine and chicken orthologs, however, are implicated in muscle differentiation and reportedly not distributed at kinetochores.We therefore conducted several assays to clarify this issue. The typical centromere staining patterns were observed in mitotic cells from both human primary culture and murine, canine, and mink cell lines. A C-terminal portion of LEK1 also conferred centromere localization. Our analysis further suggests conserved kinetochore localization of mammalian mitosin orthologs. Moreover, mitosin was associated preferentially with kinetochores of unaligned chromosomes. It was also constantly transported from kinetochores to spindle poles by cytoplasmic dynein. These properties resemble those of other kinetochore proteins important for the spindle checkpoint, thus implying a role of mitosin in this checkpoint. Therefore, mitosin family may serve as multifunctional proteins involved in both mitosis and differentiation.     

CENP-E Function at Kinetochores Is Essential for Chromosome Alignment

CENP-E is a kinesin-like protein that binds to kinetochores and may provide functions that are critical for normal chromosome motility during mitosis. To directly test the in vivo function of CENP-E, we microinjected affinity-purified antibodies to block the assembly of CENP-E onto kinetochores and then examined the behavior of these chromosomes. Chromosomes lacking CENP-E at their kinetochores consistently exhibited two types of defects that blocked their alignment at the spindle equator. Chromosomes positioned near a pole remained mono-oriented as they were unable to establish bipolar microtubule connections with the opposite pole. Chromosomes within the spindle established bipolar connections that supported oscillations and normal velocities of kinetochore movement between the poles, but these bipolar connections were defective because they failed to align the chromosomes into a metaphase plate.

Overexpression of a mutant that lacked the amino-terminal 803 amino acids of CENP-E was found to saturate limiting binding sites on kinetochores and competitively blocked endogenous CENP-E from assembling onto kinetochores. Chromosomes saturated with the truncated CENP-E mutant were never found to be aligned but accumulated at the poles or were strewn within the spindle as was the case when cells were microinjected with CENP-E antibodies. As the motor domain was contained within the portion of CENP-E that was deleted, the chromosomal defect is likely attributed to the loss of motor function.

The combined data show that CENP-E provides kinetochore functions that are essential for monopolar chromosomes to establish bipolar connections and for chromosomes with connections to both spindle poles to align at the spindle equator. Both of these events rely on activities that are provided by CENP-E's motor domain.

     

CENP-E is essential for reliable bioriented spindle attachment, but chromosome alignment can be achieved via redundant mechanisms in mammalian cells

CENP-E is a kinesin-like protein that when depleted from mammalian kinetochores leads to mitotic arrest with a mixture of aligned and unaligned chromosomes. In the present study, we used immunofluorescence, video, and electron microscopy to demonstrate that depletion of CENP-E from kinetochores via antibody microinjection reduces kinetochore microtubule binding by 23% at aligned chromosomes, and severely reduces microtubule binding at unaligned chromosomes. Disruption of CENP-E function also reduces tension across the centromere, increases the incidence of spindle pole fragmentation, and results in monooriented chromosomes approaching abnormally close to the spindle pole. Nevertheless, chromosomes show typical patterns of congression, fast poleward motion, and oscillatory motions. Furthermore, kinetochores of aligned and unaligned chromosomes exhibit normal patterns of checkpoint protein localization. These data are explained by a model in which redundant mechanisms enable kinetochore microtubule binding and checkpoint monitoring in the absence of CENP-E at kinetochores, but where reduced microtubule-binding efficiency, exacerbated by poor positioning at the spindle poles, results in chronically monooriented chromosomes and mitotic arrest. Chromosome position within the spindle appears to be a critical determinant of CENP-E function at kinetochores.     

Microtubule-dependent Changes in Assembly of Microtubule Motor Proteins and Mitotic Spindle Checkpoint Proteins at PtK1 Kinetochores

The ability of kinetochores to recruit microtubules, generate force, and activate the mitotic spindle checkpoint may all depend on microtubule- and/or tension-dependent changes in kinetochore assembly. With the use of quantitative digital imaging and immunofluorescence microscopy of PtK1 tissue cells, we find that the outer domain of the kinetochore, but not the CREST-stained inner core, exhibits three microtubule-dependent assembly states, not directly dependent on tension. First, prometaphase kinetochores with few or no kinetochore microtubules have abundant punctate or oblate fluorescence morphology when stained for outer domain motor proteins CENP-E and cytoplasmic dynein and checkpoint proteins BubR1 and Mad2. Second, microtubule depolymerization induces expansion of the kinetochore outer domain into crescent and ring morphologies around the centromere. This expansion may enhance recruitment of kinetochore microtubules, and occurs with more than a 20- to 100-fold increase in dynein and relatively little change in CENP-E, BubR1, and Mad2 in comparison to prometaphase kinetochores. Crescents disappear and dynein decreases substantially upon microtubule reassembly. Third, when kinetochores acquire their full metaphase complement of kinetochore microtubules, levels of CENP-E, dynein, and BubR1 decrease by three- to sixfold in comparison to unattached prometaphase kinetochores, but remain detectable. In contrast, Mad2 decreases by 100-fold and becomes undetectable, consistent with Mad2 being a key factor for the "wait-anaphase" signal produced by unattached kinetochores. Like previously found for Mad2, the average amounts of CENP-E, dynein, or BubR1 at metaphase kinetochores did not change with the loss of tension induced by taxol stabilization of microtubules.     

Dynein is a transient kinetochore component whose binding is regulated by microtubule attachment, not tension

Cytoplasmic dynein is the only known kinetochore protein capable of driving chromosome movement toward spindle poles. In grasshopper spermatocytes, dynein immunofluorescence staining is bright at prometaphase kinetochores and dimmer at metaphase kinetochores. We have determined that these differences in staining intensity reflect differences in amounts of dynein associated with the kinetochore. Metaphase kinetochores regain bright dynein staining if they are detached from spindle microtubules by micromanipulation and kept detached for 10 min. We show that this increase in dynein staining is not caused by the retraction or unmasking of dynein upon detachment. Thus, dynein genuinely is a transient component of spermatocyte kinetochores.We further show that microtubule attachment, not tension, regulates dynein localization at kinetochores. Dynein binding is extremely sensitive to the presence of microtubules: fewer than half the normal number of kinetochore microtubules leads to the loss of most kinetochoric dynein. As a result, the bulk of the dynein leaves the kinetochore very early in mitosis, soon after the kinetochores begin to attach to microtubules. The possible functions of this dynein fraction are therefore limited to the initial attachment and movement of chromosomes and/or to a role in the mitotic checkpoint.     

Kinetochore dynein generates a poleward pulling force to facilitate congression and full chromosome alignment

For proper chromosome segregation, all kinetochores must achieve bipolar microtubule （MT） attachment and subsequently align at the spindle equator before anaphase onset. The MT minus end-directed motor dynein/dynactin binds kinetoehores in prometaphase and has long been implicated in chromosome congression. Unfortunately, inactivation of dynein usually disturbs spindle organization, thus hampering evaluation of its kinetochore roles. Here we specifically eliminated kinetochore dynein/dynactin by RNAi-mediated depletion of ZW10, a protein essential for kinetochore localization of the motor. Time-lapse microscopy indicated markedly-reduced congression efficiency, though congressing chromosomes displayed similar velocities as in control cells. Moreover, cells frequently failed to achieve full chromosome alignment, despite their normal spindles. Confocal microcopy revealed that the misaligned kinetochores were monooriented or unattached and mostly lying outside the spindle, suggesting a difficulty to capture MTs from the opposite pole. Kinetoehores on monoastral spindles were dispersed farther away from the pole and exhibited only mild oscillation. Furthermore, inactivating dynein by other means generated similar phenotypes. Therefore, kinetochore dynein produces on monooriented kinetochores a poleward pulling force, which may contribute to efficient bipolar attachment by facilitating their proper microtubule captures to promote congression as well as full chromosome alignment.     

Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes

To understand the mechanisms which regulate meiosis-specific cell cycle and chromosome distribution in mammalian oocytes, the level and the localization of CENP-E and the kinetochore number and direction on a half bivalent were examined during pig oocyte maturation. CENP-E is a kinetochore motor protein whose intracellular level and localization are strictly regulated in the somatic cell cycle. The localizations of CENP-E on meiotic chromosomes from diakinesis stage to anaphase I and at the spindle midzone at telophase I were shown by immunofluorescent confocal microscopy to be similar to those in somatic cells of pig and other species. Further, ultrastructural analysis revealed the presence of CENP-E on fibrous corona and outer plate of kinetochores of the meiotic chromosomes. However, unlike mitosis, CENP-E staining was continuously detected either at the spindle midzone or on the kinetochores of segregated chromosomes during the first polar body emission. Consistent with this, immunoblot analysis revealed that CENP-E level remained high during meiosis I/meiosis II (MI/MII) transition and that some of CENP-E survived through the transition even in cycloheximide-treated oocytes in which cyclin B1 was completely degraded. Furthermore, examinations of CENP-E signals in confocal microscopy and kinetochores in electron microscopy in MI and MII oocytes provide the cytological evidence in mammalian oocytes which suggests that each sister chromatid in a pair has its own kinetochore which localizes side-by-side so that two sister chromatids on a half bivalent are oriented toward and connected to the same pole in MI.     

CENP-A is required for accurate chromosome segregation and sustained kinetochore association of BubR1

CENP-A is an evolutionarily conserved, centromere-specific variant of histone H3 that is thought to play a central role in directing kinetochore assembly and in centromere function. Here, we have analyzed the consequences of disrupting the CENP-A gene in the chicken DT40 cell line. In CENP-A-depleted cells, kinetochore protein assembly is impaired, as indicated by mislocalization of the inner kinetochore proteins CENP-I, CENP-H, and CENP-C as well as the outer components Nuf2/Hec1, Mad2, and CENP-E. However, BubR1 and the inner centromere protein INCENP are efficiently recruited to kinetochores. Following CENP-A depletion, chromosomes are deficient in proper congression on the mitotic spindle and there is a transient delay in prometaphase. CENP-A-depleted cells further proceed through anaphase and cytokinesis with unequal chromosome segregation, suggesting that some kinetochore function remains following substantial depletion of CENP-A. We furthermore demonstrate that CENP-A-depleted cells exhibit a specific defect in maintaining kinetochore localization of the checkpoint protein BubR1 under conditions of checkpoint activation. Our data thus point to a specific role for CENP-A in assembly of kinetochores competent in the maintenance of mitotic checkpoint signaling.     

Preventing farnesylation of the dynein adaptor Spindly contributes to the mitotic defects caused by farnesyltransferase inhibitors

The clinical interest in farnesyltransferase inhibitors (FTIs) makes it important to understand how these compounds affect cellular processes involving farnesylated proteins. Mitotic abnormalities observed after treatment with FTIs have so far been attributed to defects in the farnesylation of the outer kinetochore proteins CENP-E and CENP-F, which are involved in chromosome congression and spindle assembly checkpoint signaling. Here we identify the cytoplasmic dynein adaptor Spindly as an additional component of the outer kinetochore that is modified by farnesyltransferase (FTase). We show that farnesylation of Spindly is essential for its localization, and thus for the proper localization of dynein and its cofactor dynactin, to prometaphase kinetochores and that Spindly kinetochore recruitment is more severely affected by FTase inhibition than kinetochore recruitment of CENP-E and CENP-F. Molecular replacement experiments show that both Spindly and CENP-E farnesylation are required for efficient chromosome congression. The identification of Spindly as a new mitotic substrate of FTase provides insight into the causes of the mitotic phenotypes observed with FTase inhibitors.     

N-Terminal Polyubiquitination of the ARF Tumor Suppressor,A Natural Lysine-Less Protein

The Spindle Assembly Checkpoint ensures the fidelity of chromosome segregation at each cell division cycle. Previous reports have indicated that in higher eukaryotes checkpoint proteins, such as BubR1, are also implicated in chromosome congression, more specifically that BubR1 regulates chromosome-spindle attachments. Also, several studies have shown that BubR1 interacts with the microtubule motor protein CENP-E. Whether this association contributes to the regulation of chromosome-spindle attachments is not yet known. Accordingly, we performed a detailed analysis of microtubule-kinetochore interactions after depletion of BubR1 and the Drosophila CENP-E homologue, CENP-meta by RNAi. We find that depletion of BubR1 affects mitosis very differently from depletion of CENP-meta. While BubR1-depleted cells exit mitosis prematurely due to loss of SAC activity, CENP-meta-depleted cells accumulate in prometaphase and do not exit mitosis after spindle damage. Also, in contrast to cells depleted for CENP-meta, cells depleted for BubR1 very rarely reach full metaphase alignment even if arrested in mitosis with the proteasome inhibitor MG132. More importantly, we show for the first time that BubR1-depleted cells contain a high frequency of either monoriented or fully unattached chromosomes while most CENP-meta dsRNAi-treated cells have chromosomes attached to spindle microtubules. Moreover, simultaneous depletion of both proteins reveals that absence of CENP-meta is able to partially rescue the unattached chromosome phenotype observed after BubR1 depletion. These results strongly suggest that while BubR1 is required to promote stable microtubule kinetochore attachment, CENP-E appears to be required to destabilize kinetochore attachment. Overall our results suggest that activation of the mechanism that corrects inappropriate kinetochore attachment requires the antagonistic effects of BubR1 and CENP-E.     

NudC is required for Plk1 targeting to the kinetochore and chromosome congression

The equal distribution of chromosomes during mitosis is critical for maintaining the integrity of the genome. Essential to this process are the capture of spindle microtubules by kinetochores and the congression of chromosomes to the metaphase plate . Polo-like kinase 1 (Plk1) is a mitotic kinase that has been implicated in microtubule-kinetochore attachment, tension generation at kinetochores, tension-responsive signal transduction, and chromosome congression . The tension-sensitive substrates of Plk1 at the kinetochore are unknown. Here, we demonstrate that human Nuclear distribution protein C (NudC), a 42 kDa protein initially identified in Aspergillus nidulans and shown to be phosphorylated by Plk1 , plays a significant role in regulating kinetochore function. Plk1-phosphorylated NudC colocalizes with Plk1 at the outer plate of the kinetochore. Depletion of NudC reduced end-on microtubule attachments at kinetochores and resulted in defects in chromosome congression at the metaphase plate. Importantly, NudC-deficient cells exhibited mislocalization of Plk1 and the Kinesin-7 motor CENP-E from prometaphase kinetochores. Ectopic expression of wild-type NudC, but not NudC containing mutations in the Plk1 phosphorylation sites, recovered Plk1 localization at the kinetochore and rescued chromosome congression. Thus, NudC functions as both a substrate and a spatial regulator of Plk1 at the kinetochore to promote chromosome congression.