Background Successful human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to human oocyte maturation arrest are unknown. Methods We recruited a rare four-generation family with female infertility as a consequence of oocyte meiosis I arrest. We applied whole-exome and direct Sanger sequencing to an additional 23 patients following identification of mutations in a candidate gene, TUBB8. Expression of TUBB8 and all other β-tubulin isotypes was measured in human oocytes, early embryos, sperm cells and several somatic tissues by qRT-PCR. The effect of the TUBB8 mutations was assessed on α/β tubulin heterodimer assembly in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes via microinjection of the corresponding cRNAs. Results We identified seven mutations in the primate-specific gene TUBB8 that are responsible for human oocyte meiosis I arrest in seven families. TUBB8 expression is unique to oocytes and the early embryo, where this gene accounts for almost all of the expressed β-tubulin. The mutations affect the chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, induce microtubule chaos upon expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle assembly defects and maturation arrest upon expression in mouse and human oocytes. Conclusions TUBB8 mutations function via dominant negative effects that massively disrupt proper microtubule behavior. TUBB8 is a key gene involved in human oocyte meiotic spindle assembly and maturation.
Microtubules are essential components of axon guidance machinery. Among β-tubulin mutations, only those in TUBB3 have been shown to cause primary errors in axon guidance. All identified mutations in TUBB2B result in polymicrogyria, but it remains unclear whether TUBB2B mutations can cause axon dysinnervation as a primary phenotype. We have identified a novel inherited heterozygous missense mutation in TUBB2B that results in an E421K amino acid substitution in a family who segregates congenital fibrosis of the extraocular muscles (CFEOM) with polymicrogyria. Diffusion tensor imaging of brains of affected family members reveals aberrations in the trajectories of commissural projection neurons, implying a paucity of homotopic connections. These observations led us to ask whether axon dysinnervation is a primary phenotype, and why the E421K, but not other, TUBB2B substitutions cause CFEOM. Expression of exogenous Tubb2b-E421K in developing callosal projection neurons is sufficient to perturb homotopic connectivity, without affecting neuronal production or migration. Using in vitro biochemical assays and yeast genetics, we find that TUBB2B-E421K αβ-heterodimers are incorporated into the microtubule network where they alter microtubule dynamics and can reduce kinesin localization. These data provide evidence that TUBB2B mutations can cause primary axon dysinnervation. Interestingly, by incorporating into microtubules and altering their dynamic properties, the E421K substitution behaves differently than previously identified TUBB2B substitutions, providing mechanistic insight into the divergence between resulting phenotypes. Together with previous studies, these findings highlight that β-tubulin isotypes function in both conserved and divergent ways to support proper human nervous system development.
The cytoskeleton gives a cell its shape and plays a major role in its movement and division. It's also helps organise the content of cells and is the base for intracellular transport. Important components of the cytoskeleton are microtubules, which are hollow cylindrical beams (25 nm in diameter) that assemble from protein building blocks called tubulin. Deficiencies in microtubules are related to many diseases including cancer and Alzheimer. Given their important role, microtubules are heavily investigated in many laboratories. One way to study microtubules is to isolate them from cells and image them using light microscopy. Over the years a number of imaging techniques have been used. These techniques have a number of drawbacks which are addressed by ongoing efforts which this work is a part of. Here, we present a method based on light interference that produce high quality images of microtubules. The technique is cheap and easy to implement making it accessible to a wide base of researchers.
Summary Background To function in diverse cellular processes, the dynamic behavior of microtubules (MTs) must be differentially regulated within the cell. In budding yeast, the spindle position checkpoint (SPOC) inhibits mitotic exit in response to mispositioned spindles. To maintain SPOC-mediated anaphase arrest, astral MTs must maintain persistent interactions with and/or extend through the bud neck. However, the molecular mechanisms that ensure the stability of these interactions are not known. Results The presence of a MT extending through and/or interacting with the bud neck is maintained by spatial control of catastrophe and rescue, which extends MT lifetime >25-fold and controls the length of dynamic MTs within the bud compartment. Moreover, the single kinesin-8 motor, Kip3, alternately mediates both catastrophe and rescue of the bud MT. Kip3 accumulates in a length-dependent manner along the lattice of MTs within the bud. Yet, it induces catastrophe spatially near the bud tip. Rather, this accumulation of Kip3 facilitates its association with depolymerizing MT plus-ends, where Kip3 promotes rescue before MTs exit the bud. MT rescue within the bud requires the tail domain of Kip3, whereas the motor domain mediates catastrophe at the bud tip. In vitro, Kip3 exerts both stabilizing and destabilizing effects on reconstituted yeast MTs. Conclusions The kinesin-8 Kip3 is a multifunctional regulator that differentially stabilizes and destabilizes specific MTs. Control over MT catastrophe and rescue by Kip3 defines the length and lifetime of MTs within the bud compartment of cells with mispositioned spindles. This subcellular regulation of MT dynamics is critical to maintain mitotic arrest in response to mispositioned spindles.
We have developed and tested a robust delivery method for the transport of proteins to the cytoplasm of mammalian cells without compromising the integrity of the cell membrane. This receptormediated delivery (RMD) technology utilizes a variant of substance P (SP), a neuropeptide that is rapidly internalized upon interaction with the neurokinin-1 receptor (NK1R). Cargos in the form of synthetic antibody fragments (sABs) were conjugated to the engineered SP variant (SPv) and efficiently internalized by NK1R-expressing cells. The sABs used here were generated to bind specific conformational forms of actin. The internalized proteins appear to escape the endosome and retain their binding activity within the cells as demonstrated by co-localization with the actin cytoskeleton. Further, since the NK1R is over-expressed in many cancers, SPv-mediated delivery provides a highly specific method for therapeutic utilization of affinity reagents targeting intracellular processes in diseased tissue.actin ͉ drug delivery ͉ synthetic antibody fragment D irected delivery of bioactive reagents into cells is one of the most intensely pursued objectives in biomedical research, yet few major breakthroughs have materialized that can be broadly applied in both laboratory research and therapeutic applications. Transfection reagents (1-3) as well as cellpenetrating peptides (4-7) lack cell-type specificity, and in many cases are associated with heavy toxicity (8), restricting the successful utilization of such reagents in vivo. In contrast, receptor-mediated delivery (RMD) systems (9-11) are conceptually simple and potentially very powerful. By attaching a biomolecular cargo to a natural ligand, receptor-mediated internalization serves as an extremely efficient and selective pathway for delivery of a wide variety of bioactive entities without compromising the integrity of the cell membrane (Fig. 1). Previously described RMD methods rely on cargo attachment to hormones (12) or monoclonal antibodies that recognize a cell-surface receptor (13). However, these large protein ligands can be difficult to isolate or modify in a specific manner.Here we present a delivery method based on substance P (SP), an 11 amino acid neuropeptide that is rapidly internalized through specific interaction with the neurokinin-1 receptor (NK1R) (14-16). We have engineered a variant of SP (SPv) that enables the specific delivery of proteins to NK1R-expressing cells. Unlike larger ligands, SPv is easily chemically synthesized, allowing the incorporation of various reactive groups to facilitate coupling to myriad biomolecular cargos. Importantly, the NK1R is highly over-expressed in several aggressive tumors (17, 18), particularly astrocytomas and glioblastomas (19,20), where the level of expression is correlated with the degree of malignancy (21). Consequently, an RMD system using SPv could provide a high level of specificity for the targeting of tumor tissue, with little toxicity for noncancerous cells.To evaluate the efficacy of the SPv-NK1R delivery system, we ...
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