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PubMed2000

Numb is an endocytic protein.

Numb is a protein that in Drosophila determines cell fate as a result of its asymmetric partitioning at mitosis. The function of Numb has been linked to its ability to bind and to biologically antagonize Notch, a membrane receptor that also specifies cell fate. The biochemical mechanisms underlying the action of Numb, however, are still largely unknown. The wide pattern of expression of Numb suggests a general function in cellular homeostasis that could be additional to, or part of, its action in fate determination. Such a function could be endocytosis, as suggested by the interaction of Numb with Eps15, a component of the endocytic machinery. Here, we demonstrate that Numb is an endocytic protein. We found that Numb localizes to endocytic organelles and is cotrafficked with internalizing receptors. Moreover, it associates with the appendage domain of alpha adaptin, a subunit of AP2, a major component of clathrin-coated pits. Finally, fragments of Numb act as dominant negatives on both constitutive and ligand-regulated receptor-mediated internalization, suggesting a general role for Numb in the endocytic process.

Adaptor Protein Complex 2↗
PubMed1997

Trigeminal numbness and tic relief after microvascular decompression for typical trigeminal neuralgia.

OBJECTIVE: After most operative treatments for trigeminal neuralgia, long-term tic relief is closely correlated with postoperative numbness in the trigeminal distribution. Microvascular decompression (MVD) is proposed to relieve tic through a nontraumatic mechanism. We investigated the relationship between postoperative trigeminal numbness and tic relief in a large, prospectively followed cohort of patients treated with MVD for typical trigeminal neuralgia. METHODS: Of 1204 patients who underwent MVD for typical tic during a 20-year period, 522 had single MVDs on a single side, had not undergone ablative trigeminal procedures before or after MVD, and were still being followed in 1994. In 1994, patients graded facial numbness using a questionnaire (response rate, 92%) with a 5-point scale. Multivariate Cox and logistic regression methods were used. The analyses were adjusted for the time that had passed between the performance of MVD and the completion of the questionnaire (minimum, 2 yr). RESULTS: Seventeen percent of patients reported some degree of persistent facial numbness. Decompression of a vein at MVD (odds ratio, 2.5) and failure to find compression by the superior cerebellar artery (odds ratio, 2.0) independently predicted postoperative facial numbness, which in turn predicted postoperative burning and aching facial pain (odds ratio, 5.2-5.9). A trend toward worse outcome was noted in patients with numb faces (P = 0.3). Similar findings were noted in subgroups of patients in whom the superior cerebellar artery was decompressed at MVD (n = 381) and in whom a superior cerebellar artery with no vein was found (n = 120). In the latter subgroup, facial numbness (5.8% of patients) significantly predicted worse long-term outcome (P = 0.03). CONCLUSION: We found no evidence that postoperative trigeminal numbness predicts relief of typical tic after MVD. Trigeminal numbness was related to operative findings at MVD and predicted postoperative burning and aching facial pain. To minimize postoperative facial dysesthesia, trauma to the trigeminal root during MVD should be avoided when possible.

Decompression, Surgical↗
PubMed2002

Numb modifies neuronal vulnerability to amyloid beta-peptide in an isoform-specific manner by a mechanism involving altered calcium homeostasis: implications for neuronal death in Alzheimer's disease.

Increased production of neurotoxic forms of amyloid beta-peptide (Abeta) and abnormalities in neuronal calcium homeostasis play central roles in the pathogenesis of Alzheimer's disease (AD). Notch, a membrane receptor that controls cell-fate decisions during development of the nervous system, has been linked to AD because it is a substrate for the gamma-secretase enzyme activity that involves the presenilin-1 (PS1) protein in which mutations cause early-onset inherited AD. The actions of Notch can be antagonized by Numb, an evolutionarily conserved protein that exists in four isoforms that differ in two functional domains: a phosphotyrosine-binding (PTB) domain and a proline-rich region (PRR). We now report that Numb isoforms containing a short PTB domain increase the vulnerability of PC12 cells to death induced by Abeta1-42 and by 4-hydroxynonenal, a lipid peroxidation product previously shown to mediate neurotoxic effects of Abeta. Dysregulation of cellular calcium homeostasis occurs in cells expressing Numb isoforms with a short PTB domain, and the death-promoting effect of Numb is abolished by pharmacological inhibition of calcium release. The levels of Numb are increased in cultured primary hippocampal neurons exposed to Abeta, suggesting a role for endogenous Numb in the neuronal death process. Furthermore, higher levels of Numb were detected in the cortex of mice expressing mutant amyloid precursor protein (APP) relative to age-matched wild-type mice. Our data identify a novel isoform-specific effect of Numb on neuronal life and death cell fate decisions potentially relevant to the pathogenesis of AD. Our findings also suggest that the effects of Numb on cell fate decisions, both during development of the nervous system and in neurodegenertive disorders, are mediated by changes in cellular calcium homeostasis.

Alzheimer Disease↗
PubMed2004

Reducing lateral skin flap numbness after total knee arthroplasty.

One hundred and thirteen knees were assessed for lateral skin flap numbness, 5-227 weeks following IB2 total knee arthroplasty. The final 53 had the numb area measured. To define the anatomy of the cutaneous nerves we dissected four cadaveric knees. Eighty-six percent of patients had a numb area on objective assessment but only 60% had subjective numbness, 62% of which had improved. Patients had a larger numb area if they were aware of a numb patch, had a scar over 22 cm long or were less than 25 weeks following surgery. We conclude that not all patients will get lateral skin flap numbness and improvement occurs with time. The area of numbness can be reduced by using a shorter proximal incision, which preserves branches of the medial and intermediate cutaneous nerves of thigh.

Adult↗
PubMed1996

Asymmetric localization of a mammalian numb homolog during mouse cortical neurogenesis.

During Drosophila neurogenesis, differential segregation of Numb is necessary for daughter cells of asymmetric divisions to adopt distinct fates, at least partly by biasing the Notch-mediated cell-cell interaction. We have isolated a highly conserved mammalian homolog of Drosophila numb, m-numb. During mouse cortical neurogenesis, m-Numb is asymmetrically localized to the apical membrane of dividing ventricular neural progenitors. Depending upon the orientation of the cleavage plane, m-Numb may be distributed into one or both of the daughter cells. When expressed in Drosophila embryos, m-Numb is localized asymmetrically in dividing neural precursors and rescues the numb mutant phenotype. Furthermore, m-Numb can physically interact with mouse Notch1. We propose that some shared molecular mechanisms, both cell-intrinsic and cell-extrinsic, generate asymmetric cell divisions during neurogenesis of vertebrates and invertebrates.

Amino Acid Sequence↗
PubMed2001

Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.

Animals↗
PubMed2002

The endocytic protein alpha-Adaptin is required for numb-mediated asymmetric cell division in Drosophila.

During asymmetric cell division in Drosophila sensory organ precursor cells, the Numb protein localizes asymmetrically and segregates into one daughter cell, where it influences cell fate by repressing signal transduction via the Notch receptor. We show here that Numb acts by polarizing the distribution of alpha-Adaptin, a protein involved in receptor-mediated endocytosis. alpha-Adaptin binds to Numb and localizes asymmetrically in a Numb-dependent fashion. Mutant forms of alpha-Adaptin that no longer bind to Numb fail to localize asymmetrically and cause numb-like defects in asymmetric cell division. Our results suggest a model in which Numb influences cell fate by downregulating Notch through polarized receptor-mediated endocytosis, since Numb also binds to the intracellular domain of Notch.

Adaptor Protein Complex alpha Subunits↗
PubMed2005

Numb and alpha-Adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs.

During asymmetric cell division in Drosophila sensory organ precursors (SOPs), the Numb protein segregates into one of the two daughter cells, in which it inhibits Notch signalling to specify pIIb cell fate. We show here that Numb acts in SOP cells by inducing the endocytosis of Sanpodo, a four-pass transmembrane protein that has previously been shown to regulate Notch signalling in the central nervous system. In sanpodo mutants, SOP cells divide symmetrically into two pIIb cells. We show that Sanpodo is cortical in pIIa, but colocalizes with Notch and Delta in Rab5- and Rab7-positive endocytic vesicles in pIIb. Sanpodo endocytosis requires alpha-Adaptin, a Numb-binding partner involved in clathrin-mediated endocytosis. In numb or alpha-adaptin mutants, Sanpodo is not endocytosed. Surprisingly, this defect is observed already before and during mitosis, which suggests that Numb not only acts in pIIb, but also regulates endocytosis throughout the cell cycle. Numb binds to Sanpodo by means of its phosphotyrosine-binding domain, a region that is essential for Numb function. Our results establish numb- and alpha-adaptin-dependent endocytosis of Sanpodo as the mechanism by which Notch is regulated during external sensory organ development.

Adaptor Protein Complex alpha Subunits↗
PubMed1997

The N terminus of the Drosophila Numb protein directs membrane association and actin-dependent asymmetric localization.

Drosophila Numb is a membrane associated protein of 557 amino acids (aa) that localizes asymmetrically into a cortical crescent in mitotic neural precursor cells and segregates into one of the daughter cells, where it is required for correct cell fate specification. We demonstrate here that asymmetric localization but not membrane localization of Numb in Drosophila embryos is inhibited by latrunculin A, an inhibitor of actin assembly. We also show that deletion of either the first 41 aa or aa 41-118 of Numb eliminates both localization to the cell membrane and asymmetric localization during mitosis, whereas C-terminal deletions or deletions of central portions of Numb do not affect its subcellular localization. Fusion of the first 76 or the first 119 aa of Numb to beta-galactosidase results in a fusion protein that localizes to the cell membrane, but fails to localize asymmetrically during mitosis. In contrast, a fusion protein containing the first 227 aa of Numb and beta-galactosidase localizes asymmetrically during mitosis and segregates into the same daughter cell as the endogenous Numb protein, demonstrating that the first 227 aa of the Numb protein are sufficient for asymmetric localization.

Animals↗
PubMed1999

Characterization of four mammalian numb protein isoforms. Identification of cytoplasmic and membrane-associated variants of the phosphotyrosine binding domain.

Numb is a membrane-associated, phosphotyrosine binding (PTB) domain-containing protein that functions as an intrinsic determinant of cell fate during Drosophila development. We have identified four isoforms of mammalian Numb with predicted molecular masses of 65, 66, 71, and 72 kDa that are generated by alternative splicing of the Numb mRNA. The different isoforms result from the presence of two sequence inserts within the PTB domain and the central region of the protein. The endogenous expression pattern of these isoforms, examined using specific antisera, varied in different tissues and cell lines. In addition, differentiation of P19 cells with retinoic acid leads to the specific loss of expression of the 71- and 72-kDa Numb proteins, suggesting that the expression of certain forms of Numb protein is regulated in a cell type-specific manner. Expression of Numb proteins fused to green fluorescent protein revealed that the form of the PTB domain with the alternatively spliced insert constitutively associated with the plasma membrane in polarized Madin-Darby canine kidney cells. In contrast, the isoform without the insert was cytoplasmic, suggesting that different PTB domain isoforms may regulate the subcellular localization of Numb proteins. The membrane localization may be due, in part, to differential affinity for acidic phospholipids. The distinct expression and localization patterns of the different mammalian Numb isoforms suggest that they have distinct functional properties.

Amino Acid Sequence↗
PubMed2003

Mammalian numb proteins promote Notch1 receptor ubiquitination and degradation of the Notch1 intracellular domain.

The cell fate determinant Numb influences developmental decisions by antagonizing the Notch signaling pathway. However, the underlying molecular mechanism of this inhibition is poorly understood. Here we report that the mammalian Numb protein promotes the ubiquitination of membrane-bound Notch1 receptor. Furthermore, Numb expression resulted in the degradation of the Notch intracellular domain following activation, which correlated with a loss of Notch-dependent transcriptional activation of the Hes1 promoter as measured by a Hes1 luciferase reporter assay. The phosphotyrosine-binding (PTB) domain of Numb was required for both Notch1 ubiquitination and down-regulation of Notch1 nuclear activity. Numb-mediated ubiquitination of Notch1 was not dependent on the PEST region, which was previously shown to mediate Sel10-dependent ubiquitination of Notch in the nucleus, suggesting a distinct E3 ubiquitin ligase is involved. In agreement we demonstrate that Numb interacts with the cytosolic HECT domain-containing E3 ligase Itch and that Numb and Itch act cooperatively to promote ubiquitination of membrane-tethered Notch1. These results suggest that Numb recruits components of the ubiquitination machinery to the Notch receptor thereby facilitating Notch1 ubiquitination at the membrane, which in turn promotes degradation of the intracellular domain circumventing its nuclear translocation and downstream activation of Notch1 target genes.

3T3 Cells↗
PubMed2006

The enigma of the numb-Notch relationship during mammalian embryogenesis.

Asymmetric cell division is an attractive means to diversify cell fates during development and for stem cells to balance self-renewal and differentiation. In Drosophila, two signaling pathways, one mediated by Numb and the other by Notch, play essential but antagonistic roles in enabling the two daughters to adopt different fates after a wide variety of asymmetric cell divisions. However, recent studies show that mutating mammalian Numb homologues, m-Numb and Numblike (Numbl or Nbl), and eliminating Notch signaling in the developing nervous system both lead to premature depletion of neural stem/progenitor cells in mice. These findings raise an interesting question as to whether and how the antagonistic roles of Numb and Notch signaling are conserved in vertebrates. Here we provide evidence that loss of m-Numb and Numbl outside the embryonic nervous system also causes phenotypes similar to those exhibited by mice with defective Notch signaling. We further show that very little Numb protein is necessary for embryogenesis and that the presence of different m-Numb isoforms is unlikely to provide a molecular basis for differential regulation of Notch signaling in mammals, as these isoforms are functionally indistinguishable in cell fate specification. We discuss possible mechanisms by which the antagonistic roles of Numb and Notch are evolutionarily conserved to meet the needs of stem cell maintenance during mammalian neurogenesis.

Animals↗
PubMed2005

The Notch regulator Numb links the Notch and TCR signaling pathways.

Both the Notch and TCR signaling pathways play an important role in T cell development, but the links between these signaling pathways are largely unexplored. The adapter protein Numb is a well-characterized inhibitor of Notch and also contains a phosphotyrosine binding domain, suggesting that Numb could provide a link between these pathways. We explored this possibility by investigating the physical interactions among Notch, Numb, and the TCR signaling apparatus and by examining the consequences of a Numb mutation on T cell development. We found that Notch and Numb cocluster with the TCR at the APC contact during Ag-driven T cell-APC interactions in both immature and mature T cells. Furthermore, Numb coimmunoprecipitates with components of the TCR signaling apparatus. Despite this association, T cell development and T cell activation occur normally in the absence of Numb, perhaps due to the expression of the related protein, Numblike. Together our data suggest that Notch and TCR signals may be integrated at the cell membrane, and that Numb may be an important adapter in this process.

Animals↗
PubMed2026

Tumoral switch in NUMB splicing changes essential transcription pathways and induces malignant properties in tumour cells.

BACKGROUND: Emerging evidence indicates that cancer is associated with widespread splicing alterations that generate tumour-specific isoforms. One example is NUMB, an evolutionarily conserved adaptor protein, which produces four isoforms (p72, p71, p66, and p65) through alternative splicing of exons 3 and 9. Although traditionally considered as a tumour suppressor, NUMB has also been reported as an oncogene. We propose that this dual role reflects isoform-specific expression. RESULTS: Using public databases, we identify a tumour-associated switch in NUMB isoform expression: p72 and p71 are upregulated in tumours, whereas p66 and p65 are more highly expressed in non-tumour tissues. These isoforms show distinct associations with key cellular processes. NUMBL, a NUMB homolog, displays expression patterns similar to p65. We further identify two transcriptional clusters: one characterised by high expression of p72 and p71, and the other by enhanced p66/p65/NUMBL expression. These clusters exhibit differential associations with Notch, WNT/β-catenin, Hedgehog, and Hippo signalling pathways, suggesting isoform-specific regulatory roles. In breast cancer cell lines, we develop a NUMB-score based on isoform expression, which classifies cell lines into biologically distinct groups. The p72/p71-enriched group shows distinct signatures, pathway activity, and drug sensitivity. Application of this score to TCGA-BRCA samples reveals a significant link between high NUMB-score and poor survival, as confirmed by Kaplan-Meier analysis. CONCLUSIONS: We find that NUMB emerges as a potential oncogenic contributor and biomarker in the context of splicing-based precision oncology, highlighting Isoform-specific expression as a clinical determinant of tumour behaviour, pathway activity, and therapeutic response.

Humans↗
PubMed2005

Localization-dependent and -independent roles of numb contribute to cell-fate specification in Drosophila.

During asymmetric cell division, protein determinants are segregated into one of the two daughter cells. The Numb protein acts as a segregating determinant during both mouse and Drosophila development. In flies, Numb localizes asymmetrically and is required for cell-fate specification in the central and peripheral nervous systems, as well as during muscle and heart development. Whether its asymmetric segregation is important to the performance of these functions is not firmly established. Here, we demonstrate that Numb acts both in a localization-dependent and in a localization-independent manner. We have generated numb mutants that affect only the asymmetric localization of the protein during mitosis. We demonstrate that asymmetric segregation of Numb into one of the two daughter cells is absolutely essential for cell-fate specification in the Drosophila peripheral nervous system. Numb localization is also essential in MP2 neuroblasts in the central nervous system and during muscle development. Surprisingly, in dividing ganglion mother cells or during heart development, Numb function is independent of its ability to segregate asymmetrically in mitosis. Our results suggest that two classes of asymmetric cell division exist, each with different requirements for asymmetric inheritance of cell-fate determinants.

Animals↗
PubMed2006

Phosphorylation of Numb regulates its interaction with the clathrin-associated adaptor AP-2.

Numb is thought to participate in clathrin-dependent endocytosis by directly interacting with the clathrin-associated adaptor complex AP-2, although the underlying mechanisms are unknown. Numb is also known to be phosphorylated at Ser(264)in vitro and in vivo. Here, we found that Numb is phosphorylated in vitro by Ca(2+)/calmodulin-dependent protein kinase I on Ser(283). This phosphorylation was also observed in transfected COS-7 cells, indicating its physiological relevance. Pull-down experiments showed that the phosphorylation of Numb impaired its binding to the AP-2 complex and simultaneously recruited 14-3-3 proteins in vitro. Based on experiments using Numb mutants, both the initial phosphorylation of Ser(264) and the subsequent phosphorylation of Ser(283) are sufficient to abolish the binding of Numb to AP-2 and to promote the interaction with 14-3-3 protein. These findings suggest a novel mechanism for the regulation of Numb-mediated endocytosis, namely through direct phosphorylation.

Adaptor Protein Complex 2↗
PubMed2006

Role of relaxation and specific suggestions in hypnotic emotional numbing.

The cognitive characteristics of highly hypnotizable subjects (Highs) allow them to easily modify their cognitive and autonomic state. Under hypnosis, Highs receiving cognitive, fear-like stimulation exhibit the cardiovascular changes typical of fear/stress, but also show an EEG pattern indicating a balance between fear-induced arousal and hypnotic relaxation. Indeed, hypnosis is effective in the attenuation of both emotional experience and behaviour (emotional numbing). The aim of the present experiment was to investigate the possible different role of relaxation and suggestion in hypnotic emotional numbing. Tonic skin conductance, respirogram, heart rate, systolic and diastolic blood pressure were recorded in 3 groups of hypnotized subjects: Group 1 received a fearful guided imagery associated with threat suggestions (Threat) followed by the same fearful suggestion associated with numbing instructions (relaxation and "No-Threat"); Group 2 received the same instructions in the opposite order of presentation; Group 3 received the fearful suggestion with threat instructions twice. The numbing suggestion reduced fear-related emotional experience and autonomic responses; if No-Threat preceded Threat, the heart rate, heart rate variability and blood pressure were also reduced during Threat, in spite of self reports of high negative emotion. Thus, 1) the subjective experience and the autonomic response to fear can be dissociated; 2) the efficacy of numbing suggestion is extended to a subsequent Threat stimulation; 3) habituation does not contribute to the numbing effect. The results indicate that the specific numbing suggestion is the main factor in hypnotic modulation of the experience of fear.

Adult↗
PubMed2004

Murine numb regulates granule cell maturation in the cerebellum.

Notch is a key regulator of vertebrate neurogenesis and the cytoplasmic adaptor protein Numb is a modulator of the Notch signaling pathway. To address the role of murine Numb in development of the central nervous system, we used a conditional gene ablation approach. We show that Numb is involved in the maturation of cerebellar granule cells. Although the specification of neural cell fates in the cerebellum is not affected in the absence of Numb, the transition from a mitotic progenitor to a mature granule cell is aberrant and migration of postmitotic granule cells to the internal granule cell layer is delayed. In some animals, this results in a complete agenesis of granule cells and a strong ataxia. We confirmed these findings in vitro and found that Numb-deficient cerebellar progenitor cells show a marked delay in granule cell maturation. Our results suggest that Numb plays a role in the transition of a mitotic progenitor to a fully differentiated granule cell in the cerebellum. In addition, the maturation of Purkinje cells is also delayed in Numb-deficient mice.

Animals↗