Δ3-tubulin impairs mitotic spindle morphology and increases nuclear size in pancreatic cancer cells.

Cancer cell proliferation is affected by post-translational modifications of tubulin. Especially, overexpression or depletion of enzymes for modifications on the tubulin C-terminal region perturbs dynamic instability of the spindle body. Those modifications include processing of C-terminal amino acids of α-tubulin; detyrosination, and a removal of penultimate glutamic acid (Δ2). We previously found a further removal of the third last glutamic acid, which generates so-called Δ3-tubulin. The effects of Δ3-tubulin on spindle integrities and cell proliferation remain to be elucidated. In this study, we investigated the impacts of forced expression of Δ3-tubulin on the structure of spindle bodies and cell division in a pancreatic cancer cell line, PANC-1. Overexpression of HA-tagged Δ3-tubulin impaired the morphology and orientation of spindle bodies during cell division in PANC-1 cells. In particular, spindle bending was most significantly increased. Expression of EGFP-tagged Δ3-tubulin driven by the endogenous promoter of human TUBA1B also deformed and misoriented spindle bodies. Spindle bending and condensation defects were significantly observed by EGFP-Δ3-tubulin expression. Furthermore, EGFP-Δ3-tubulin expression increased the nuclear size in a dose-dependent manner of EGFP-Δ3-tubulin expression. The expression of EGFP-Δ3-tubulin tended to slow down cell proliferation. Taken together, our results demonstrate that Δ3-tubulin affects the spindle integrity and cell division.

Circadian oscillation in primary cilium length by clock genes regulates fibroblast cell migration.

Various mammalian cells have autonomous cellular clocks that are produced by the transcriptional cycle of clock genes. Cellular clocks provide circadian rhythms for cellular functions via transcriptional and cytoskeletal regulation. The vast majority of mammalian cells possess a primary cilium, an organelle protruding from the cell surface. Here, we investigated the little-known relationship between circadian rhythm and primary cilia. The length and number of primary cilia showed circadian dynamics both in vitro and in vivo. The circadian rhythm of primary cilium length was abolished by SR9011 and Bmal1 knockout. A centrosomal protein, pericentrin, transiently accumulates in centriolar satellites, the base of primary cilia at the shortest cilia phase, and induces elongation of primary cilia at the longest cilia phase in the circadian rhythm of primary cilia. In addition, rhythmic cell migration during wound healing depends on the length of primary cilia and affects the rate of wound healing. Our findings demonstrate that the circadian dynamics of primary cilium length by clock genes control fibroblast migration and could provide new insights into chronobiology.

Time-lapse imaging of primary cilium behavior with physiological expression of fluorescent ciliary proteins.

Almost all cell types of mammals have a small protrusion named a primary cilium on their surface. Primary cilia are enriched by cilia-specific ion channels and G-protein-coupled receptors. They are known to regulate various cellular functions that contribute to the development and homeostasis of living organisms by receiving extracellular signals and transfusing them to the cell body. All functions are performed when the structure of the primary cilia is maintained properly. Abnormalities in primary cilia or their signaling can lead to a collection of diseases in various organs called ciliopathies. The primary cilium is dynamic, static, or fixed. The length of primary cilia varies as the cell cycle progresses and is also altered by extracellular stimuli. Ligand binding to cilia-specific receptors is also known to alter the length. Thus, there is a need for a method to study the morphological changes of the primary cilium in a time-dependent manner, especially under stimuli or mechanical shocks. Time-lapse imaging of primary cilia is one of the most powerful methods to capture the time-dependent behavior of primary cilia. Overexpression of ciliary proteins fused to fluorescent proteins is commonly used for the time-lapse imaging of primary cilia. However, overexpression has drawbacks in terms of artifacts. In addition, the time-lapse imaging of the tiny primary cilia requires some technical tricks. Here, we present a detailed description of the methods for time-lapse imaging of primary cilium, from the generation of cell lines that stably express fluorescent protein-labeled cilia-localized proteins at the physiological level to image analysis, including quantification through image acquisition.

A pair of primers facing at the double-strand break site enables to detect NHEJ-mediated indel mutations at a 1-bp resolution.

The introduction of small insertion/deletion (indel) mutations in the coding region of genes by the site-specific nucleases such as Cas9 allows researchers to obtain frameshift null mutants. Technically simple and costly reasonable genotyping methods are awaited to efficiently screen the frameshift null mutant candidates. Here, we developed a simple genotyping method called DST-PCR (Double-strand break Site-Targeted PCR) using "face-to-face" primers where the 3' ends of forward and reverse primers face each other at the position between 3-bp and 4-bp upstream of the PAM sequence, which is generally the Cas9-mediated double-strand break site. Generated amplicons are directly subjected to TBE-High-Resolution PAGE, which contains a high concentration of bis-acrylamide, for mutant clones detection with 1-bp resolution. We present actual cases of screening of CRISPR/Cas9-engineered knockout (KO) cells for six genes, where we screen indels to obtain potential KO cell clones utilizing our approach. This method allowed us to detect 1-bp to 2-bp insertion and 1-bp to 4-bp deletion in one or both alleles of mutant cell clones. In addition, this technique also allowed the identification of heterozygous and homozygous biallelic functional KO candidates. Thus, DST-PCR is a simple and fast method to screen KO candidates generated by the CRISPR/Cas9 system before the final selection of clones with sequencing.

Knock-in of Labeled Proteins into 5'UTR Enables Highly Efficient Generation of Stable Cell Lines.

Stable cell lines and animal models expressing tagged proteins are important tools for studying behaviors of cells and molecules. Several molecular biology technologies have been applied with varying degrees of success and efficiencies to establish cell lines expressing tagged proteins. Here we applied CRISPR/Cas9 for the knock-in of tagged proteins into the 5'UTR of the endogenous gene loci. With this 5'UTR-targeting knock-in strategy, stable cell lines expressing Arl13b-Venus, Reep6-HA, and EGFP-alpha-tubulin were established with high efficiencies ranging from 50 to 80% in antibiotic selected cells. The localization of the knock-in proteins were identical to that of the endogenous proteins in wild-type cells and showed homogenous expression. Moreover, the expression of knock-in EGFP-alpha-tubulin from the endogenous promoter was stable over long-term culture. We further demonstrated that the fluorescent signals were enough for a long time time-lapse imaging. The fluorescent signals were distinctly visible during the whole duration of the time-lapse imaging and showed specific subcellular localizations. Altogether, our strategy demonstrates that 5'UTR is an amenable site to generate cell lines for the stable expression of tagged proteins from endogenous loci in mammalian cells.Key words: CRISPR/Cas9, knock-in, primary cilium, UTR, tubulin.

Current understandings of the relationship between extracellular vesicles and cilia.

Mammalian cells have a tiny hair-like protrusion on their surface called a primary cilium. Primary cilia are thought to be the antennae for the cells, receiving signals from the environment. In some studies, extracellular vesicles (EVs) were found attached to the surface of the primary cilium. An idea for the phenomenon is that the primary cilium is the receptor for receiving the EVs. Meanwhile, a unicellular organism, Chlamydomonas, which has two long cilia, usually called flagella, release EVs termed ectosomes from the surface of the flagella. Accumulating evidence suggests that the primary cilium also functions as the 'emitter' of EVs. Physiological and pathological impacts are also elucidated for the release of EVs from primary cilia. However, the roles of released cilia-derived EVs remain to be clarified. This review introduces the historical background of the relationship between EVs and cilia, and recent progresses in the research field.

Live cell imaging of dynamic behaviors of motile cilia and primary cilium.

The cilium is a tiny organelle, with a length of 1-10 µm and a diameter of ~200 nm, that projects from the surface of many cells and functions to generate fluid flow and/or sense extracellular signals from the environment. Abnormalities in cilia may cause a broad spectrum of disease, i.e. the so-called ciliopathies. Multiple imaging approaches have been implemented to understand the structure, motion and function of the tiny cilium. In this review, we focus on the microscopic observations and analyses of the dynamic behaviors of both motile cilia and primary cilium. Motile cilia repeat reciprocal motions at 15-25 Hz with a clear asymmetry of effective and recovery strokes. Observing the fast movement of motile cilia requires a high-speed camera with a frame rate of more than 100 fps. The labeling of cilia tips enables the detailed analysis of the asymmetric beating motion of motile cilia. The primary cilium, which is imagined to be 'static,' is also dynamic, i.e. it elongates, shrinks and disassembles, although this behavior is quite slower than that of motile cilia. The specific fluorescent labeling of primary cilium and time-lapse imaging are required to observe and analyze the slow behaviors of the primary cilium. We present some approaches, including some tips for successful procedures, in the successful imaging of the dynamic behaviors of motile cilia and primary cilium.

Proper cytoskeletal architecture beneath the plasma membrane of red blood cells requires Ttll4.

Mammalian red blood cells (RBCs) circulate through blood vessels, including capillaries, for tens of days under high mechanical stress. RBCs tolerate this mechanical stress while maintaining their shape because of their elastic membrane skeleton. This membrane skeleton consists of spectrin-actin lattices arranged as quasi-hexagonal units beneath the plasma membrane. In this study, we found that the organization of the RBC cytoskeleton requires tubulin tyrosine ligase-like 4 (Ttll4). RBCs from Ttll4-knockout mice showed larger average diameters in smear test. Based on the rate of hemolysis, Ttll4-knockout RBCs showed greater vulnerability to phenylhydrazine-induced oxidative stress than did wild-type RBCs. Ultrastructural analyses revealed the macromolecular aggregation of cytoskeletal components in RBCs of Ttll4-knockout mice. Immunoprecipitation using the anti-glutamylation antibody GT335 revealed nucleosome assembly protein 1 (NAP1) to be the sole target of TTLL4 in the RBCs, and NAP1 glutamylation was completely lost in Ttll4-knockout RBCs. In wild-type RBCs, the amount of glutamylated NAP1 in the membrane was nearly double that in the cytosol. Furthermore, the absence of TTLL4-dependent glutamylation of NAP1 weakened the binding of NAP1 to the RBC membrane. Taken together, these data demonstrate that Ttll4 is required for proper cytoskeletal organization in RBCs.