Truncated variants of MAGEL2 are involved in the etiologies of the Schaaf-Yang and Prader-Willi syndromes.

The neurodevelopmental disorders Prader-Willi syndrome (PWS) and Schaaf-Yang syndrome (SYS) both arise from genomic alterations within human chromosome 15q11-q13. A deletion of the SNORD116 cluster, encoding small nucleolar RNAs, or frameshift mutations within MAGEL2 result in closely related phenotypes in individuals with PWS or SYS, respectively. By investigation of their subcellular localization, we observed that in contrast to a predominant cytoplasmic localization of wild-type (WT) MAGEL2, a truncated MAGEL2 mutant was evenly distributed between the cytoplasm and the nucleus. To elucidate regulatory pathways that may underlie both diseases, we identified protein interaction partners for WT or mutant MAGEL2, in particular the survival motor neuron protein (SMN), involved in spinal muscular atrophy, and the fragile-X-messenger ribonucleoprotein (FMRP), involved in autism spectrum disorders. The interactome of the non-coding RNA SNORD116 was also investigated by RNA-CoIP. We show that WT and truncated MAGEL2 were both involved in RNA metabolism, while regulation of transcription was mainly observed for WT MAGEL2. Hence, we investigated the influence of MAGEL2 mutations on the expression of genes from the PWS locus, including the SNORD116 cluster. Thereby, we provide evidence for MAGEL2 mutants decreasing the expression of SNORD116, SNORD115, and SNORD109A, as well as protein-coding genes MKRN3 and SNRPN, thus bridging the gap between PWS and SYS.

Chronic spindle assembly checkpoint activation causes myelosuppression and gastrointestinal atrophy.

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor, CDC20, preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However, the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression, we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for, gastrointestinal atrophy is detrimental. Remarkably, deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome, while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect, identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast, only overexpression of anti-apoptotic BCL2, but none of the BH3-only protein deficiencies mentioned above, can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo.

Adaptations of the 3T3-L1 adipocyte lipidome to defective ether lipid catabolism upon Agmo knockdown.

Little is known about the physiological role of alkylglycerol monooxygenase (AGMO), the only enzyme capable of cleaving the 1-O-alkyl ether bond of ether lipids. Expression and enzymatic activity of this enzyme can be detected in a variety of tissues including adipose tissue. This labile lipolytic membrane-bound protein uses tetrahydrobiopterin as a cofactor, and mice with reduced tetrahydrobiopterin levels have alterations in body fat distribution and blood lipid concentrations. In addition, manipulation of AGMO in macrophages led to significant changes in the cellular lipidome, and alkylglycerolipids, the preferred substrates of AGMO, were shown to accumulate in mature adipocytes. Here, we investigated the roles of AGMO in lipid metabolism by studying 3T3-L1 adipogenesis. AGMO activity was induced over 11 days using an adipocyte differentiation protocol. We show that RNA interference-mediated knockdown of AGMO did not interfere with adipocyte differentiation or affect lipid droplet formation. Furthermore, lipidomics revealed that plasmalogen phospholipids were preferentially accumulated upon Agmo knockdown, and a significant shift toward longer and more polyunsaturated acyl side chains of diacylglycerols and triacylglycerols could be detected by mass spectrometry. Our results indicate that alkylglycerol catabolism has an influence not only on ether-linked species but also on the degree of unsaturation in the massive amounts of triacylglycerols formed during in vitro 3T3-L1 adipocyte differentiation.

A VSV-based assay quantifies coronavirus Mpro/3CLpro/Nsp5 main protease activity and chemical inhibition.

Protease inhibitors are among the most powerful antiviral drugs. However, for SARS-CoV-2 only a small number of protease inhibitors have been identified thus far and there is still a great need for assays that efficiently report protease activity and inhibition in living cells. Here, we engineer a safe VSV-based system to report both gain- and loss-of-function of coronavirus main protease (Mpro/3CLpro/Nsp5) activity in living cells. We use SARS-CoV-2 3CLpro in this system to confirm susceptibility to known inhibitors (boceprevir, GC376, PF-00835231, and PF-07321332/nirmatrelvir) and reevaluate other reported inhibitors (baicalein, ebselen, carmofur, ethacridine, ivermectin, masitinib, darunavir, and atazanavir). Moreover, we show that the system can be adapted to report both the function and the chemical inhibition of proteases from different coronavirus species as well as from distantly related viruses. Together with the fact that live cell assays also reflect compound permeability and toxicity, we anticipate that this system will be useful for both identification and optimization of additional coronavirus protease inhibitors.

Genetic and functional evidence for gp130/IL6ST-induced transient receptor potential ankyrin 1 upregulation in uninjured but not injured neurons in a mouse model of neuropathic pain.

ABSTRACT: Peripheral nerve injuries result in pronounced alterations in dorsal root ganglia, which can lead to the development of neuropathic pain. Although the polymodal mechanosensitive transient receptor potential ankyrin 1 (TRPA1) ion channel is emerging as a relevant target for potential analgesic therapies, preclinical studies do not provide unequivocal mechanistic insight into its relevance for neuropathic pain pathogenesis. By using a transgenic mouse model with a conditional depletion of the interleukin-6 (IL-6) signal transducer gp130 in Nav1.8 expressing neurons (SNS-gp130-/-), we provide a mechanistic regulatory link between IL-6/gp130 and TRPA1 in the spared nerve injury (SNI) model. Spared nerve injury mice developed profound mechanical hypersensitivity as indicated by decreased withdrawal thresholds in the von Frey behavioral test in vivo, as well as a significant increase in mechanosensitivity of unmyelinated nociceptive primary afferents in ex vivo skin-nerve recordings. In contrast to wild type and control gp130fl/fl animals, SNS-gp130-/- mice did not develop mechanical hypersensitivity after SNI and exhibited low levels of Trpa1 mRNA in sensory neurons, which were partially restored by adenoviral gp130 re-expression in vitro. Importantly, uninjured but not injured neurons developed increased responsiveness to the TRPA1 agonist cinnamaldehyde, and neurons derived from SNS-gp130-/- mice after SNI were significantly less responsive to cinnamaldehyde. Our study shows for the first time that TRPA1 upregulation is attributed specifically to uninjured neurons in the SNI model, and this depended on the IL-6 signal transducer gp130. We provide a solution to the enigma of TRPA1 regulation after nerve injury and stress its significance as an important target for neuropathic pain disorders.

Feedback control of the Gpr161-Gαs-PKA axis contributes to basal Hedgehog repression in zebrafish.

Hedgehog (Hh) ligands act as morphogens to direct patterning and proliferation during embryonic development. Protein kinase A (PKA) is a central negative regulator of Hh signalling, and in the absence of Hh ligands, PKA activity prevents inappropriate expression of Hh target genes. The orphan G-protein-coupled receptor Gpr161 contributes to the basal Hh repression machinery by activating PKA. Gpr161 acts as an A-kinase-anchoring protein, and is itself phosphorylated by PKA, but the functional significance of PKA phosphorylation of Gpr161 in the context of Hh signalling remains unknown. Here, we show that loss of Gpr161 in zebrafish leads to constitutive activation of medium and low, but not maximal, levels of Hh target gene expression. Furthermore, we find that PKA phosphorylation-deficient forms of Gpr161, which we show directly couple to Gαs, display an increased sensitivity to Shh, resulting in excess high-level Hh signalling. Our results suggest that PKA feedback-mediated phosphorylation of Gpr161 may provide a mechanism for fine-tuning Gpr161 ciliary localisation and PKA activity.

Phosphorylation of Drosophila CENP-A on serine 20 regulates protein turn-over and centromere-specific loading.

Centromeres are specialized chromosomal regions epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A is required for kinetochore formation which is essential for chromosome segregation during mitosis. Spatial restriction of CENP-A to the centromere is tightly controlled. Its overexpression results in ectopic incorporation and the formation of potentially deleterious neocentromeres in yeast, flies and in various human cancers. While the contribution of posttranslational modifications of CENP-A to these processes has been studied in yeast and mammals to some extent, very little is known about Drosophila melanogaster. Here, we show that CENP-A is phosphorylated at serine 20 (S20) by casein kinase II and that in mitotic cells, the phosphorylated form is enriched on chromatin. Importantly, our results reveal that S20 phosphorylation regulates the turn-over of prenucleosomal CENP-A by the SCFPpa-proteasome pathway and that phosphorylation promotes removal of CENP-A from ectopic but not from centromeric sites in chromatin. We provide multiple lines of evidence for a crucial role of S20 phosphorylation in controlling restricted incorporation of CENP-A into centromeric chromatin in flies. Modulation of the phosphorylation state of S20 may provide the cells with a means to fine-tune CENP-A levels in order to prevent deleterious loading to extra-centromeric sites.

Biogenesis of lysosome-related organelles complex-1 (BORC) regulates late endosomal/lysosomal size through PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate.

Mechanisms that control lysosomal function are essential for cellular homeostasis. Lysosomes adapt in size and number to cellular needs but little is known about the underlying molecular mechanism. We demonstrate that the late endosomal/lysosomal multimeric BLOC-1-related complex (BORC) regulates the size of these organelles via PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2 ] production. Deletion of the core BORC component Diaskedin led to increased levels of PI(3,5)P2 , suggesting activation of PIKfyve, and resulted in enhanced lysosomal reformation and subsequent reduction in lysosomal size. This process required AMP-activated protein kinase (AMPK), a known PIKfyve activator, and was additionally dependent on the late endosomal/lysosomal adaptor, mitogen-activated protein kinases and mechanistic target of rapamycin activator (LAMTOR/Ragulator) complex. Consistently, in response to glucose limitation, AMPK activated PIKfyve, which induced lysosomal reformation with increased baseline autophagy and was coupled to a decrease in lysosomal size. These adaptations of the late endosomal/lysosomal system reversed under glucose replete growth conditions. In summary, our results demonstrate that BORC regulates lysosomal reformation and size in response to glucose availability.

Subcellular Localization of Sprouty2 in Human Glioma Cells.

Sprouty proteins act ubiquitously as signaling integrators and inhibitors of receptor tyrosine kinase (RTK) activated pathways. Among the four Sprouty isoforms, Sprouty2 is a key regulator of growth factor signaling in several neurological disorders. High protein levels correlate with reduced survival of glioma patients. We recently demonstrated that abrogating its function inhibits tumor growth by overstimulation of ERK and induction of DNA replication stress. The important role of Sprouty2 in the proliferation of malignant glioma cells prompted us to investigate its subcellular localization applying super-resolution fluorescence and immunoelectron microscopy. We found that cytoplasmic Sprouty2 is not homogenously distributed but localized to small spots (<100 nm) partly attached to vimentin filaments and co-localized with activated ERK. The protein is associated with early, late and recycling endosomes in response to but also independently of growth factor stimulation. The subcellular localization of Sprouty2 in all areas exhibiting strong RTK activities may reflect a protective response of glioma cells to limit excessive ERK activation and to prevent cellular senescence and apoptosis.

Sprouty2 enhances the tumorigenic potential of glioblastoma cells.

Background: Sprouty2 (SPRY2), a feedback regulator of receptor tyrosine kinase (RTK) signaling, has been shown to be associated with drug resistance and cell proliferation in glioblastoma (GBM), but the underlying mechanisms are still poorly defined. Methods: SPRY2 expression and survival patterns of patients with gliomas were analyzed using publicly available databases. Effects of RNA interference targeting SPRY2 on cellular proliferation in established GBM or patient-derived GBM stemlike cells were examined. Loss- or gain-of-function of SPRY2 to regulate the tumorigenic capacity was assessed in both intracranial and subcutaneous xenografts. Results: SPRY2 was found to be upregulated in GBM, which correlated with reduced survival in GBM patients. SPRY2 knockdown significantly impaired proliferation of GBM cells but not of normal astrocytes. Silencing of SPRY2 increased epidermal growth factor-induced extracellular signal-regulated kinase (ERK) and Akt activation causing premature onset of DNA replication, increased DNA damage, and impaired proliferation, suggesting that SPRY2 suppresses DNA replication stress. Abrogating SPRY2 function strongly inhibited intracranial tumor growth and led to significantly prolonged survival of U87 xenograft-bearing mice. In contrast, SPRY2 overexpression promoted tumor propagation of low-tumorigenic U251 cells. Conclusions: The present study highlights an antitumoral effect of SPRY2 inhibition that is based on excessive activation of ERK signaling and DNA damage response, resulting in reduced cell proliferation and increased cytotoxicity, proposing SPRY2 as a promising pharmacological target in GBM patients.

Checkpoint kinase 1 is essential for normal B cell development and lymphomagenesis.

Checkpoint kinase 1 (CHK1) is critical for intrinsic cell cycle control and coordination of cell cycle progression in response to DNA damage. Despite its essential function, CHK1 has been identified as a target to kill cancer cells and studies using Chk1 haploinsufficient mice initially suggested a role as tumor suppressor. Here, we report on the key role of CHK1 in normal B-cell development, lymphomagenesis and cell survival. Chemical CHK1 inhibition induces BCL2-regulated apoptosis in primary as well as malignant B-cells and CHK1 expression levels control the timing of lymphomagenesis in mice. Moreover, total ablation of Chk1 in B-cells arrests their development at the pro-B cell stage, a block that, surprisingly, cannot be overcome by inhibition of mitochondrial apoptosis, as cell cycle arrest is initiated as an alternative fate to limit the spread of damaged DNA. Our findings define CHK1 as essential in B-cell development and potent target to treat blood cancer.

Transient apoptosis inhibition in donor stem cells improves hematopoietic stem cell transplantation.

During hematopoietic stem cell transplantation, a substantial number of donor cells are lost because of apoptotic cell death. Transplantation-associated apoptosis is mediated mainly by the proapoptotic BCL-2 family proteins BIM and BMF, and their proapoptotic function is conserved between mouse and human stem and progenitor cells. Permanent inhibition of apoptosis in donor cells caused by the loss of these BH3-only proteins improves transplantation outcome, but recipients might be exposed to increased risk of lymphomagenesis or autoimmunity. Here, we address whether transient inhibition of apoptosis can serve as a safe but efficient alternative to improve the outcome of stem cell transplantation. We show that transient apoptosis inhibition by short-term overexpression of prosurvival BCL-XL, known to block BIM and BMF, is not only sufficient to increase the viability of hematopoietic stem and progenitor cells during engraftment but also improves transplantation outcome without signs of adverse pathologies. Hence, this strategy represents a promising and novel therapeutic approach, particularly under conditions of limited donor stem cell availability.

Citron Kinase Deficiency Leads to Chromosomal Instability and TP53-Sensitive Microcephaly.

Mutations in citron (CIT), leading to loss or inactivation of the citron kinase protein (CITK), cause primary microcephaly in humans and rodents, associated with cytokinesis failure and apoptosis in neural progenitors. We show that CITK loss induces DNA damage accumulation and chromosomal instability in both mammals and Drosophila. CITK-deficient cells display "spontaneous" DNA damage, increased sensitivity to ionizing radiation, and defective recovery from radiation-induced DNA lesions. In CITK-deficient cells, DNA double-strand breaks increase independently of cytokinesis failure. Recruitment of RAD51 to DNA damage foci is compromised by CITK loss, and CITK physically interacts with RAD51, suggesting an involvement of CITK in homologous recombination. Consistent with this scenario, in doubly CitK and Trp53 mutant mice, neural progenitor cell death is dramatically reduced; moreover, clinical and neuroanatomical phenotypes are remarkably improved. Our results underscore a crucial role of CIT in the maintenance of genomic integrity during brain development.

The PIDDosome activates p53 in response to supernumerary centrosomes.

Centrosomes, the main microtubule-organizing centers in animal cells, are replicated exactly once during the cell division cycle to form the poles of the mitotic spindle. Supernumerary centrosomes can lead to aberrant cell division and have been causally linked to chromosomal instability and cancer. Here, we report that an increase in the number of mature centrosomes, generated by disrupting cytokinesis or forcing centrosome overduplication, triggers the activation of the PIDDosome multiprotein complex, leading to Caspase-2-mediated MDM2 cleavage, p53 stabilization, and p21-dependent cell cycle arrest. This pathway also restrains the extent of developmentally scheduled polyploidization by regulating p53 levels in hepatocytes during liver organogenesis. Taken together, the PIDDosome acts as a first barrier, engaging p53 to halt the proliferation of cells carrying more than one mature centrosome to maintain genome integrity.

Both IGF1R and INSR Knockdown Exert Antitumorigenic Effects in Prostate Cancer In Vitro and In Vivo.

The IGF network with its main receptors IGF receptor 1 (IGF1R) and insulin receptor (INSR) is of major importance for cancer initiation and progression. To date, clinical studies targeting this network were disappointing and call for thorough analysis of the IGF network in cancer models. We highlight the oncogenic effects controlled by IGF1R and INSR in prostate cancer cells and show similarities as well as differences after receptor knockdown (KD). In PC3 prostate cancer cells stably transduced with inducible short hairpin RNAs, targeting IGF1R or INSR attenuated cell growth and proliferation ultimately driving cells into apoptosis. IGF1R KD triggered rapid and strong antiproliferative and proapoptotic responses, whereas these effects were less pronounced and delayed after INSR KD. Down-regulation of the antiapoptotic proteins myeloid cell leukemia-1 and survivin was observed in both KDs, whereas IGF1R KD also attenuated expression of prosurvival proteins B cell lymphoma-2 and B cell lymphoma-xL. Receptor KD induced cell death involved autophagy in particular upon IGF1R KD; however, no difference in mitochondrial energy metabolism was observed. In a mouse xenograft model, induction of IGF1R or INSR KD after tumor establishment eradicated most of the tumors. After 20 days of receptor KD, tumor cells were found only in 1/14 IGF1R and 3/14 INSR KD tumor remnants. Collectively, our data underline the oncogenic functions of IGF1R and INSR in prostate cancer namely growth, proliferation, and survival in vitro as well as in vivo and identify myeloid cell leukemia-1 and survivin as important mediators of inhibitory and apoptotic effects.

The NOXA-MCL1-BIM axis defines lifespan on extended mitotic arrest.

Cell death on extended mitotic arrest is considered arguably most critical for the efficacy of microtubule-targeting agents (MTAs) in anticancer therapy. While the molecular machinery controlling mitotic arrest on MTA treatment, the spindle assembly checkpoint (SAC), appears well defined, the molecular components executing cell death, as well as factors connecting both networks remain poorly understood. Here we conduct a mini screen exploring systematically the contribution of individual BCL2 family proteins at single cell resolution to death on extended mitotic arrest, and demonstrate that the mitotic phosphorylation of BCL2 and BCLX represent a priming event for apoptosis that is ultimately triggered by NOXA-dependent MCL1 degradation, enabling BIM-dependent cell death. Our findings provide a comprehensive model for the initiation of apoptosis in cells stalled in mitosis and provide a molecular basis for the increased efficacy of combinatorial treatment of cancer cells using MTAs and BH3 mimetics.

Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome.

Tetrahydrobiopterin is a cofactor synthesized from GTP with well-known roles in enzymatic nitric oxide synthesis and aromatic amino acid hydroxylation. It is used to treat mild forms of phenylketonuria. Less is known about the role of tetrahydrobiopterin in lipid metabolism, although it is essential for irreversible ether lipid cleavage by alkylglycerol monooxygenase. Here we found intracellular alkylglycerol monooxygenase activity to be an important regulator of alkylglycerol metabolism in intact murine RAW264.7 macrophage-like cells. Alkylglycerol monooxygenase was expressed and active also in primary mouse bone marrow-derived monocytes and "alternatively activated" M2 macrophages obtained by interleukin 4 treatment, but almost missing in M1 macrophages obtained by IFN-γ and lipopolysaccharide treatment. The cellular lipidome of RAW264.7 was markedly changed in a parallel way by modulation of alkylglycerol monooxygenase expression and of tetrahydrobiopterin biosynthesis affecting not only various ether lipid species upstream of alkylglycerol monooxygenase but also other more complex lipids including glycosylated ceramides and cardiolipins, which have no direct connection to ether lipid pathways. Alkylglycerol monooxygenase activity manipulation modulated the IFN-γ/lipopolysaccharide-induced expression of inducible nitric oxide synthase, interleukin-1ß, and interleukin 1 receptor antagonist but not transforming growth factor ß1, suggesting that alkylglycerol monooxygenase activity affects IFN-γ/lipopolysaccharide signaling. Our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in ether lipid metabolism of murine macrophages and reveal that alteration of alkylglycerol monooxygenase activity has a profound impact on the lipidome also beyond the class of ether lipids.

Statin-induced depletion of geranylgeranyl pyrophosphate inhibits cell proliferation by a novel pathway of Skp2 degradation.

Statins, such as lovastatin, can induce a cell cycle arrest in the G1 phase. This robust antiproliferative activity remains intact in many cancer cells that are deficient in cell cycle checkpoints and leads to an increased expression of CDK inhibitor proteins p27Kip1 and p21Cip1. The molecular details of this statin-induced growth arrest remains unclear. Here we present evidence that lovastatin can induce the degradation of Skp2, a subunit of the SCFSkp2 ubiquitin ligase that targets p27Kip1 and p21Cip1 for proteasomal destruction. The statin-induced degradation of Skp2 is cell cycle phase independent and does not require its well characterised degradation pathway mediated by APC/CCdh1- or Skp2 autoubiquitination. An N-terminal domain preceding the F-box of Skp2 is both necessary and sufficient for its statin mediated degradation. The degradation of Skp2 results from statin induced depletion of geranylgeranyl isoprenoid intermediates of cholesterol biosynthesis. Inhibition of geranylgeranyl-transferase-I also promotes APC/CCdh1- independent degradation of Skp2, indicating that de-modification of a geranylgeranylated protein triggers this novel pathway of Skp2 degradation.

PIAS1 is a crucial factor for prostate cancer cell survival and a valid target in docetaxel resistant cells.

Occurrence of an inherent or acquired resistance to the chemotherapeutic drug docetaxel is a major burden for patients suffering from different kinds of malignancies, including castration resistant prostate cancer (PCa). In the present study we address the question whether PIAS1 targeting can be used to establish a basis for improved PCa treatment. The expression status and functional relevance of PIAS1 was evaluated in primary tumors, in metastatic lesions, in tissue of patients after docetaxel chemotherapy, and in docetaxel resistant cells. Patient data were complemented by functional studies on PIAS1 knockdown in vitro as well as in chicken chorioallantoic membrane and mouse xenograft in vivo models. PIAS1 was found to be overexpressed in local and metastatic PCa and its expression was further elevated in tumors after docetaxel treatment as well as in docetaxel resistant cells. Furthermore, PIAS1 knockdown experiments revealed an increased expression of tumor suppressor p21 and declined expression of anti-apoptotic protein Mcl1, which caused diminished cell proliferation and tumor growth in vitro and in vivo. In summary, the presented data indicate that PIAS1 is crucial for parental and docetaxel resistant PCa cell survival and is therefore a promising new target for treatment of primary, metastatic, and chemotherapy resistant PCa.

Kinetochore motors drive congression of peripheral polar chromosomes by overcoming random arm-ejection forces.

Accurate chromosome segregation during cell division in metazoans relies on proper chromosome congression at the equator. Chromosome congression is achieved after bi-orientation to both spindle poles shortly after nuclear envelope breakdown, or by the coordinated action of motor proteins that slide misaligned chromosomes along pre-existing spindle microtubules. These proteins include the minus-end-directed kinetochore motor dynein, and the plus-end-directed motors CENP-E at kinetochores and chromokinesins on chromosome arms. However, how these opposite and spatially distinct activities are coordinated to drive chromosome congression remains unknown. Here we used RNAi, chemical inhibition, kinetochore tracking and laser microsurgery to uncover the functional hierarchy between kinetochore and arm-associated motors, exclusively required for congression of peripheral polar chromosomes in human cells. We show that dynein poleward force counteracts chromokinesins to prevent stabilization of immature/incorrect end-on kinetochore-microtubule attachments and random ejection of polar chromosomes. At the poles, CENP-E becomes dominant over dynein and chromokinesins to bias chromosome ejection towards the equator. Thus, dynein and CENP-E at kinetochores drive congression of peripheral polar chromosomes by preventing arm-ejection forces mediated by chromokinesins from working in the wrong direction.