FlexiBAC: a versatile, open-source baculovirus vector system for protein expression, secretion, and proteolytic processing.

BACKGROUND: Baculovirus-mediated expression in insect cells is a powerful approach for protein production. However, many existing methods are time-consuming, offer limited options for protein tagging, and are unsuitable for secreted proteins requiring proteolytic maturation, such as TGF-ß family growth factors. RESULTS: To overcome the limitations of traditional baculovirus expression systems, we engineered "FlexiBAC". This system allows recombinant baculovirus formation inside insect cells and reduces the time between initial cloning and protein production to 13 days. FlexiBAC includes 143 shuttle vectors that append combinations of purification tags, fluorescent markers, proteolytic cleavage sites, trafficking signals, and chemical conjugation tags to the termini of the target protein. This system also overexpresses recombinant furin convertase to allow efficient proteolytic processing of secreted proteins. We demonstrate that FlexiBAC can be used to produce high levels of mature, active forms of TGF-ß family growth factors, such as Activin A, as well as other proteins that are typically difficult to reconstitute, such as proteins rich in coiled-coil, low complexity, and disordered domains. CONCLUSIONS: FlexiBAC is a protein expression system for production of both cytosolic proteins and secreted proteins that require proteolytic maturation. The design of FlexiBAC and its expansive complementary shuttle vector system reduces cloning steps and simplifies baculovirus production.

Pseudotyped baculovirus is an effective gene expression tool for studying molecular function during axolotl limb regeneration.

Axolotls can regenerate complex structures through recruitment and remodeling of cells within mature tissues. Accessing the underlying mechanisms at a molecular resolution is crucial to understand how injury triggers regeneration and how it proceeds. However, gene transformation in adult tissues can be challenging. Here we characterize the use of pseudotyped baculovirus (BV) as an effective gene transfer method both for cells within mature limb tissue and within the blastema. These cells remain competent to participate in regeneration after transduction. We further characterize the effectiveness of BV for gene overexpression studies by overexpressing Shh in the blastema, which yields a high penetrance of classic polydactyly phenotypes. Overall, our work establishes BV as a powerful tool to access gene function in axolotl limb regeneration.

Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration.

Salamanders exhibit extensive regenerative capacities and serve as a unique model in regeneration research. However, due to the lack of targeted gene knockin approaches, it has been difficult to label and manipulate some of the cell populations that are crucial for understanding the mechanisms underlying regeneration. Here we have established highly efficient gene knockin approaches in the axolotl (Ambystoma mexicanum) based on the CRISPR/Cas9 technology. Using a homology-independent method, we successfully inserted both the Cherry reporter gene and a larger membrane-tagged Cherry-ERT2-Cre-ERT2 (∼5-kb) cassette into axolotl Sox2 and Pax7 genomic loci. Depending on the size of the DNA fragments for integration, 5-15% of the F0 transgenic axolotl are positive for the transgene. Using these techniques, we have labeled and traced the PAX7-positive satellite cells as a major source contributing to myogenesis during axolotl limb regeneration. Our work brings a key genetic tool to molecular and cellular studies of axolotl regeneration.

A tunable refractive index matching medium for live imaging cells, tissues and model organisms.

In light microscopy, refractive index mismatches between media and sample cause spherical aberrations that often limit penetration depth and resolution. Optical clearing techniques can alleviate these mismatches, but they are so far limited to fixed samples. We present Iodixanol as a non-toxic medium supplement that allows refractive index matching in live specimens and thus substantially improves image quality in live-imaged primary cell cultures, planarians, zebrafish and human cerebral organoids.

Serum Proteases Potentiate BMP-Induced Cell Cycle Re-entry of Dedifferentiating Muscle Cells during Newt Limb Regeneration.

Limb amputation in the newt induces myofibers to dedifferentiate and re-enter the cell cycle to generate proliferative myogenic precursors in the regeneration blastema. Here we show that bone morphogenetic proteins (BMPs) and mature BMPs that have been further cleaved by serum proteases induce cell cycle entry by dedifferentiating newt muscle cells. Protease-activated BMP4/7 heterodimers that are present in serum strongly induced myotube cell cycle re-entry with protease cleavage yielding a 30-fold potency increase of BMP4/7 compared with canonical BMP4/7. Inhibition of BMP signaling via muscle-specific dominant-negative receptor expression reduced cell cycle entry in vitro and in vivo. In vivo inhibition of serine protease activity depressed cell cycle re-entry, which in turn was rescued by cleaved-mimic BMP. This work identifies a mechanism of BMP activation that generates blastema cells from differentiated muscle.

One-step purification of assembly-competent tubulin from diverse eukaryotic sources.

We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from Saccharomyces cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions. The eluted tubulin is fully functional and can be efficiently assembled into microtubules. The method eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research.

Purification of tubulin from porcine brain.

Microtubules, polymers of the heterodimeric protein αß-tubulin, give shape to cells and are the tracks for vesicle transport and chromosome segregation. In vitro assays to study microtubule functions and their regulation by microtubule-associated proteins require the availability of purified αß-tubulin. In this chapter, we describe the process of purification of heterodimeric αß-tubulin from porcine brain.

Synergy of glucose and growth hormone signalling in islet cells through ICA512 and STAT5.

Nutrients and growth hormones promote insulin production and the proliferation of pancreatic beta-cells. An imbalance between ever-increasing metabolic demands and insulin output causes diabetes. Recent evidence indicates that beta-cells enhance insulin gene expression depending on their secretory activity. This signalling pathway involves a catalytically inactive receptor tyrosine phosphatase, ICA512, whose cytoplasmic tail is cleaved on glucose-stimulated exocytosis of insulin secretory granules and then moves into the nucleus, where it upregulates insulin transcription. Here, we show that the cleaved cytosolic fragment of ICA512 enhances the transcription of secretory granule genes (including its own gene) by binding to tyrosine phosphorylated signal transducers and activators of transcription (STAT) 5 and preventing its dephosphorylation. Sumoylation of ICA512 by the E3 SUMO ligase PIASy, in turn, may reverse this process by decreasing the binding of ICA512 to STAT5. These findings illustrate how the exocytosis of secretory granules, through a retrograde pathway that sustains STAT activity, converges with growth hormone signalling to induce adaptive changes in beta-cells in response to metabolic demands.

Global and local control of microtubule destabilization promoted by a catastrophe kinesin MCAK/XKCM1.

Traditionally, kinesins have been identified as proteins that use the energy of ATP to translocate along microtubules. However, in the last decade some kinesin-like proteins were found to destabilize microtubule ends. The kinesins that destabilize microtubules are known as "catastrophe kinesins". Analyses of a Xenopus member of the catastrophe kinesins called MCAK/XKCM1 have shown that, in fact, catastrophe kinesins are essential for controlling the distribution of microtubules by inducing their depolymerization. Therefore, unraveling the mechanisms of how microtubule destabilization promoted by these catastrophe kinesins is controlled is essential for understanding how microtubules in a cell are distributed. Here we give an overview of the studies that have focused on the global and local control of microtubule destabilization promoted by MCAK/XKCM1.

Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis.

Centrosomes act as sites of microtubule growth, but little is known about how the number and stability of microtubules emanating from a centrosome are controlled during the cell cycle. We studied the role of the TACC3-XMAP215 complex in this process by using purified proteins and Xenopus laevis egg extracts. We show that TACC3 forms a one-to-one complex with and enhances the microtubule-stabilizing activity of XMAP215 in vitro. TACC3 enhances the number of microtubules emanating from mitotic centrosomes, and its targeting to centrosomes is regulated by Aurora A-dependent phosphorylation. We propose that Aurora A regulation of TACC3 activity defines a centrosome-specific mechanism for regulation of microtubule polymerization in mitosis.

A comparison of the ability of XMAP215 and tau to inhibit the microtubule destabilizing activity of XKCM1.

During mitosis, microtubules not only grow fast, but also have a high rate of catastrophe. This is achieved in part by the activity of the MAP, XMAP215, which can stimulate the growth rate of microtubules without fully inhibiting the function of the catastrophe-kinesin XKCM1. We do not know whether this activity is particular to XMAP215, or is a general property of all MAPs. Here, we compare the activities of XMAP215 with the neuronal MAP tau, in opposing the destabilizing activity of the non-conventional kinesin XKCM1. We show that tau is a much more potent inhibitor of XKCM1 than XMAP215. Because tau completely suppresses XKCM1 activity, even at low concentrations, the combination of tau and XKCM1 is unable to generate mitotic microtubule dynamics.

Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization.

Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150(Glued) are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH(2) terminus of p150(Glued) binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150(Glued) and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH(2)-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH(2) and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.

Plasticity and reprogramming of differentiated cells in amphibian regeneration: partial purification of a serum factor that triggers cell cycle re-entry in differentiated muscle cells.

The reversal of cellular differentiation to form proliferating progenitor cells is a critical aspect of regenerative ability in the urodele amphibians. This process has been studied using skeletal muscle during limb or tail regeneration, or dorsal iris epithelium during lens regeneration. An unknown activity in serum triggers cell cycle re-entry from the differentiated state. Here we describe the biochemical properties and fractionation of this serum factor. The factor is a glycoprotein that associates with large molecular weight complexes. The purification and molecular identification of the serum factor represents an important avenue in understanding regenerative ability and dedifferentiation capacity on a molecular basis.

Transfer of scrapie prion infectivity by cell contact in culture.

BACKGROUND: When a cell is infected with scrapie prions, newly synthesized molecules of the prion protein PrP(C) are expressed at the cell surface and may subsequently be converted to the abnormal form PrP(Sc). In an experimental scrapie infection of an animal, the initial innoculum of PrP(Sc) is cleared relatively rapidly, and the subsequent propagation of the infection depends on the ability of infected cells to convert uninfected target cells to stable production of PrP(Sc). The mechanism of such cell-based infection is not understood. RESULTS: We have established a system in dissociated cell culture in which scrapie-infected mouse SMB cells are able to stably convert genetically marked target cells by coculture. After coculture and rigorous removal of SMB cells, the target cells express PrP(Sc) and also incorporate [35S]methionine into PrP(Sc). The extent of conversion was sensitive to the ratio of the two cell types, and conversion by live SMB required 2500-fold less PrP(Sc) than conversion by a cell-free prion preparation. The conversion activity of SMB cells is not detectable in conditioned medium and apparently depends on close proximity or contact, as evidenced by culturing the SMB and target cells on neighboring but separate surfaces. SMB cells were killed by fixation in aldehydes, followed by washing, and were found to retain significant activity at conversion of target cells. CONCLUSIONS: Cell-mediated infection of target cells in this culture system is effective and requires significantly less PrP(Sc) than infection by a prion preparation. Several lines of evidence indicate that it depends on cell contact, in particular, the activity of aldehyde-fixed infected cells.