50 Years of Image Analysis

Microtubules are core components of the eukaryotic cytoskeleton with essential roles in cell division, shaping, motility and intracellular transport. Despite their functional heterogeneity, microtubules have a highly conserved structure made from almost identical molecular building blocks: the tubulin proteins. Alternative tubulin isotypes and a variety of post-translational modifications control the properties and functions of the microtubule cytoskeleton, a concept known as the 'tubulin code'. Here we review the current understanding of the molecular components of the tubulin code and how they impact microtubule properties and functions. We discuss how tubulin isotypes and post-translational modifications control microtubule behaviour at the molecular level and how this translates into physiological functions at the cellular and organism levels. We then go on to show how fine-tuning of microtubule function by some tubulin modifications can affect homeostasis and how perturbation of this fine-tuning can lead to a range of dysfunctions, many of which are linked to human disease.

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The tubulin code and its role in controlling microtubule properties and functions.

Infrared photodetectors are currently subject to a rapidly expanding application space, with an increasing demand for compact, sensitive and inexpensive detectors. Despite continued advancement, technological factors limit the widespread usage of such detectors, specifically, the need for cooling and the high costs associated with processing of iii–v/ii–vi semiconductors. Here, black phosphorous (bP)/MoS2 heterojunction photodiodes are explored as mid-wave infrared (MWIR) detectors. Although previous studies have demonstrated photodiodes using bP, here we significantly improve the performance, showing that such devices can be competitive with conventional MWIR photodetectors. By optimizing the device structure and light management, we demonstrate a two-terminal device that achieves room-temperature external quantum efficiencies (ηe) of 35% and specific detectivities (D*) as high as 1.1 × 1010 cm Hz1/2 W−1 in the MWIR region. Furthermore, by leveraging the anisotropic optical properties of bP we demonstrate the first bias-selectable polarization-resolved photodetector that operates without the need for external optics.

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Polarization-resolved black phosphorus/molybdenum disulfide mid-wave infrared photodiodes with high detectivity at room temperature

Biological processes are naturally regulated with high spatial and temporal control, as is perhaps most evident in metazoan embryogenesis Chemical tools have been extensively utilized in cell and developmental biology to investigate cellular processes, and conditional control methods have expanded applications of these technologies toward resolving complex biological questions Light represents an excellent external trigger since it can be controlled with very high spatial and temporal precision To this end, several optically regulated tools have been developed and applied to living systems In this review we discuss recent developments of optochemical tools, including small molecules, peptides, proteins, and nucleic acids that can be irreversibly or reversibly controlled through light irradiation, with a focus on applications in cells and animals

Optochemical Control of Biological Processes in Cells and Animals.

Black phosphorus is an infrared layered material. Its bandgap complements other widely studied two-dimensional materials: zero-gap graphene and visible/near-infrared gap transition metal dichalcogenides. Although highly desirable, a comprehensive infrared characterization is still lacking. Here we report a systematic infrared study of mechanically exfoliated few-layer black phosphorus, with thickness ranging from 2 to 15 layers and photon energy spanning from 0.25 to 1.36 eV. Each few-layer black phosphorus exhibits a thickness-dependent unique infrared spectrum with a series of absorption resonances, which reveals the underlying electronic structure evolution and serves as its infrared fingerprints. Surprisingly, unexpected absorption features, which are associated with the forbidden optical transitions, have been observed. Furthermore, we unambiguously demonstrate that controllable uniaxial strain can be used as a convenient and effective approach to tune the electronic structure of few-layer black phosphorus. Our study paves the way for black phosphorus applications in infrared photonics and optoelectronics. Few-layered black phosphorus offers an infrared bandgap, complementing that of graphene and transition metal dichalcogenides. Here, the authors investigate the thickness- and strain-dependent electronic structure of black phosphorus using polarised infrared spectroscopy.

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Infrared fingerprints of few-layer black phosphorus.

Genetic elements compete for transmission through meiosis, when haploid gametes are created from a diploid parent. Selfish elements can enhance their transmission through a process known as meiotic drive. In female meiosis, selfish elements drive by preferentially attaching to the egg side of the spindle. This implies some asymmetry between the two sides of the spindle, but the molecular mechanisms underlying spindle asymmetry are unknown. Here we found that CDC42 signaling from the cell cortex regulated microtubule tyrosination to induce spindle asymmetry and that non-Mendelian segregation depended on this asymmetry. Cortical CDC42 depends on polarization directed by chromosomes, which are positioned near the cortex to allow the asymmetric cell division. Thus, selfish meiotic drivers exploit the asymmetry inherent in female meiosis to bias their transmission.

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Spindle asymmetry drives non-Mendelian chromosome segregation

Protein localization in cells can yield much information about the spatial arrangement of cellular processes and the participating groups. Here, the authors present a membrane-permeable and photoactive agent for localized protein dimerization in cells.

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Localized light-induced protein dimerization in living cells using a photocaged dimerizer

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Strategies for selectively increasing O-GlcNAc levels on a target protein in cells would accelerate studies of this essential modification. Here, we report a generalizable strategy for introducing O-GlcNAc into selected target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT yielded nanobody-OGT constructs that selectively delivered O-GlcNAc to a series of tagged target proteins (e.g., JunB, cJun, and Nup62). Truncation of the tetratricopeptide repeat domain as in OGT(4) increased selectivity for the target protein through the nanobody by reducing global elevation of O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein JunB and Nup62. Finally, we demonstrate the ability to selectively target endogenous α-synuclein for O-GlcNAcylation in HEK293T cells. These first proximity-directed OGT constructs provide a flexible strategy for targeting additional proteins and a template for further engineering of OGT and the O-GlcNAc proteome in the future. The use of a nanobody to redirect OGT substrate selection for glycosylation of desired proteins in cells may further constitute a generalizable strategy for controlling a broader array of post-translational modifications in cells.

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Engineering a Proximity-Directed O-GlcNAc Transferase for Selective Protein O-GlcNAcylation in Cells.

Kinetochores act as hubs for multiple activities during cell division, including microtubule interactions and spindle checkpoint signaling. Each kinetochore can act autonomously, and activities change rapidly as proteins are recruited to, or removed from, kinetochores. Understanding this dynamic system requires tools that can manipulate kinetochores on biologically relevant temporal and spatial scales. Optogenetic approaches have the potential to provide temporal and spatial control with molecular specificity. Here we report new chemical inducers of protein dimerization that allow us to both recruit proteins to and release them from kinetochores using light. We use these dimerizers to manipulate checkpoint signaling and molecular motor activity. Our findings demonstrate specialized properties of the CENP-E (kinesin-7) motor for directional chromosome transport to the spindle equator and for maintenance of metaphase alignment. This work establishes a foundation for optogenetic control of kinetochore function, which is broadly applicable to experimental probing of other dynamic cellular processes.

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Optogenetic control of kinetochore function.

O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions.

Chanat Aonbangkhen

Papers

Spindle asymmetry drives non-Mendelian chromosome segregation

Localized light-induced protein dimerization in living cells using a photocaged dimerizer

Engineering a Proximity-Directed O-GlcNAc Transferase for Selective Protein O-GlcNAcylation in Cells.

Optogenetic control of kinetochore function.

Aspartate Residues Far from the Active Site Drive O-GlcNAc Transferase Substrate Selection.