Label-free high-speed wide-field imaging of single microtubules using interference reflection microscopy.

When studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labelling or there is concern that labelling will disrupt function. Commonly used techniques for observing unlabelled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has a similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high-speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the optical quality of interference reflection microscopy falls within the range of other microscope techniques, being inferior to some and superior to others, depending on the metric used and, with minimal microscope modification, can be used to study the dynamics of unlabelled microtubules. LAY DESCRIPTION: 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.

Mutations in TUBB8 and Human Oocyte Meiotic Arrest.

Background 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 the arrest of human oocyte maturation are unknown. Methods We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other ß-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one ß-tubulin polypeptide (α/ß-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. Results We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed ß-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/ß-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. Conclusions TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility. (Funded by the National Basic Research Program of China and others.).

An inherited TUBB2B mutation alters a kinesin-binding site and causes polymicrogyria, CFEOM and axon dysinnervation.

Microtubules are essential components of axon guidance machinery. Among ß-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 αß-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 ß-tubulin isotypes function in both conserved and divergent ways to support proper human nervous system development.

An engineered substance P variant for receptor-mediated delivery of synthetic antibodies into tumor cells.

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 receptor-mediated 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.