Skin substitutes with noncultured autologous skin cell suspension heal porcine full-thickness wounds in a one-stage procedure.

Clinical application of skin substitute is typically a two-stage procedure with application of skin substitute matrix to the wound followed by engraftment of a split-thickness skin graft (STSG). This two-stage procedure requires multiple interventions, increasing the time until the wound is epithelialised. In this study, the feasibility of a one-stage procedure by combining bioengineered collagen-chondroitin-6-sulfate (DS1) or decellularised fetal bovine skin substitute (DS2) with autologous skin cell suspension (ASCS) in a porcine full-thickness wound healing model was evaluated. Twelve full-thickness excisional wounds on the backs of pigs received one of six different treatments: empty; ASCS; DS1 with or without ASCS; DS2 with or without ASCS. The ASCS was prepared using a point-of-care device and was seeded onto the bottom side of DS1, DS2, and empty wounds at 80 000 cells/cm2 . Wound measurements and photographs were taken on days 0, 9, 14, 21, 28, 35, and 42 post-wounding. Histological analysis was performed on samples obtained on days 9, 14, 28, and 42. Wounds in the empty group or with ASCS alone showed increased wound contraction, fibrosis, and myofibroblast density compared with other treatment groups. The addition of ASCS to DS1 or DS2 resulted in a marked increase in re-epithelialisation of wounds at 14 days, from 15 ± 11% to 71 ± 20% (DS1 vs DS1 + ASCS) or 28 ± 14% to 77 ± 26 (DS2 vs DS2 + ASCS) despite different mechanisms of tissue regeneration employed by the DS used. These results suggest that this approach may be a viable one-stage treatment in clinical practice.

Verifying the function and localization of genetically encoded Ca2+ sensors and converting FRET ratios to Ca2+ concentrations.

Genetically encoded, ratiometric, fluorescent Ca(2+) biosensors can be used in living cells to quantitatively measure free Ca(2+) concentrations in the cytosol or in organelles. This protocol describes how to perform a calibration of a Ca(2+) sensor expressed in cultured mammalian cells as images are acquired using a widefield fluorescence microscope. This protocol also explains how to calculate Förster resonance energy transfer (FRET) ratios from acquired images and how to convert FRET ratios to Ca(2+) concentrations.

Measuring the in situ Kd of a genetically encoded Ca2+ sensor.

The use of genetically encoded Ca(2+) sensors (GECIs) for long-term monitoring of intracellular Ca(2+) has become increasingly common in the last decade. Emission-ratiometric GECIs, such as those in the Yellow Cameleon family, can be used to make quantitative measurements, meaning that their fluorescence signals can be converted to free Ca(2+) concentrations ([Ca(2+)]free). This conversion is only as accurate as the sensor's apparent dissociation constant for Ca(2+) (K'd), which depends on temperature, pH, and salt concentration. This protocol describes a method for performing a titration, in living cells (in situ), of cytosolic, nuclear, or mitochondrial sensors.

Properties and use of genetically encoded FRET sensors for cytosolic and organellar Ca2+ measurements.

In the last 15 years, there has been an explosion in the development of genetically encoded biosensors that report enzyme activity, chemical transformation, or concentration of ions and molecules in living cells. Currently, there are well over 120 biosensors of different cellular targets. As a general design principle, these sensors convert a molecular event, such as the binding of a molecule to a sensing domain or a signal-induced change in protein conformation, into a change in the sensor's fluorescence properties. In contrast to small-molecule sensors, genetically encoded sensors are generated when sensor-encoding nucleic acid sequences, which have been introduced by transgenic technologies, are translated in cells, tissues, or organisms. One of the best developed classes of biosensors is the genetically encoded Ca(2+) indicators (GECIs). Here, we briefly summarize the properties of ratiometric GECIs and describe how they are used to quantify Ca(2+) in specific cellular locations, such as the cytosol, nucleus, endoplasmic reticulum, and mitochondria.

Quantitative measurement of Ca2+ and Zn2+ in mammalian cells using genetically encoded fluorescent biosensors.

Genetically encoded, ratiometric, fluorescent biosensors can be used to quantitatively measure intracellular ion concentrations in living cells. We describe important factors to consider when selecting a Ca(2+) or Zn(2+) biosensor, such as the sensor's dissociation constant (K(d')) and its dynamic range. We also discuss the limits of quantitative measurement using these sensors and reasons why a sensor may perform differently in different biological systems or subcellular compartments. We outline protocols for (1) quickly confirming sensor functionality in a new biological system, (2) calibrating a sensor to convert a sensor's FRET ratio to ion concentration, and (3) titrating a sensor in living cells to obtain its K(d') under different experimental conditions.

Promotion of vesicular zinc efflux by ZIP13 and its implications for spondylocheiro dysplastic Ehlers-Danlos syndrome.

Zinc is essential but potentially toxic, so intracellular zinc levels are tightly controlled. A key strategy used by many organisms to buffer cytosolic zinc is to store it within vesicles and organelles.It is yet unknown whether vesicular or organellar sites perform this function in mammals. Human ZIP13, a member of the Zrt/Irt-like protein (ZIP) metal transporter family, might provide an answer to this question. Mutations in the ZIP13 gene, SLC39A13, previously were found to cause the spondylocheiro dysplastic form of Ehlers­Danlos syndrome (SCD-EDS), a heritable connective tissue disorder.Those previous studies suggested that ZIP13 transports excess zinc out of the early secretory pathway and that zinc overload in the endoplasmic reticulum (ER) occurs in SCD-EDS patients. In contrast,this study indicates that ZIP13's role is to release labile zinc from vesicular stores for use in the ER and other compartments. We propose that SCD-EDS is the result of vesicular zinc trapping and ER zinc deficiency rather than overload.

New alternately colored FRET sensors for simultaneous monitoring of Zn²âº in multiple cellular locations.

Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for reporting on ions, molecules and biochemical reactions in living cells. Here we describe the development of new sensors for Zn²âºbased on alternate FRET-pairs that do not involve the traditional CFP and YFP. Zn²âº is an essential micronutrient and plays fundamental roles in cell biology. Consequently there is a pressing need for robust sensors to monitor Zn²âº levels and dynamics in cells with high spatial and temporal resolution. Here we develop a suite of sensors using alternate FRET pairs, including tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE. These sensors were targeted to both the nucleus and cytosol and characterized and validated in living cells. Sensors based on the new FRET pair Clover/mRuby2 displayed a higher dynamic range and better signal-to-noise ratio than the remaining sensors tested and were optimal for monitoring changes in cytosolic and nuclear Zn²âº. Using a green-red sensor targeted to the nucleus and cyan-yellow sensor targeted to either the ER, Golgi, or mitochondria, we were able to monitor Zn²âº uptake simultaneously in two compartments, revealing that nuclear Zn²âº rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn²âº more slowly and with a delay of 600-700 sec. Lastly, these studies provide the first glimpse of nuclear Zn²âº and reveal that nuclear Zn²âº is buffered at a higher level than cytosolic Zn²âº.

New sensors for quantitative measurement of mitochondrial Zn(2+).

Zinc (Zn(2+)) homeostasis plays a vital role in cell function, and the dysregulation of intracellular Zn(2+) is associated with mitochondrial dysfunction. Few tools exist to quantitatively monitor the buffered, free Zn(2+) concentration in mitochondria of living cells ([Zn(2+)](mito)). We have validated three high dynamic range, ratiometric, genetically encoded, fluorescent Zn(2+) sensors that we have successfully used to precisely measure and monitor [Zn(2+)](mito) in several cell types. Using one of these sensors, called mito-ZapCY1, we report observations that free Zn(2+) is buffered at concentrations about 3 orders of magnitude lower in mitochondria than in the cytosol and that HeLa cells expressing mito-ZapCY1 have an average [Zn(2+)](mito) of 0.14 pM, which differs significantly from other cell types. These optimized mitochondrial Zn(2+) sensors could improve our understanding of the relationship between Zn(2+) homeostasis and mitochondrial function.

Measuring steady-state and dynamic endoplasmic reticulum and Golgi Zn2+ with genetically encoded sensors.

Zn(2+) plays essential roles in biology, and cells have adopted exquisite mechanisms for regulating steady-state Zn(2+) levels. Although much is known about total Zn(2+) in cells, very little is known about its subcellular distribution. Yet defining the location of Zn(2+) and how it changes with signaling events is essential for elucidating how cells regulate this essential ion. Here we create fluorescent sensors genetically targeted to the endoplasmic reticulum (ER) and Golgi to monitor steady-state Zn(2+) levels as well as flux of Zn(2+) into and out of these organelles. These studies reveal that ER and Golgi contain a concentration of free Zn(2+) that is 100 times lower than the cytosol. Both organelles take up Zn(2+) when cytosolic levels are elevated, suggesting that the ER and Golgi can sequester elevated cytosolic Zn(2+) and thus have the potential to play a role in influencing Zn(2+) toxicity. ER Zn(2+) homeostasis is perturbed by small molecule antagonists of Ca(2+) homeostasis and ER Zn(2+) is released upon elevation of cytosolic Ca(2+) pointing to potential exchange of these two ions across the ER. This study provides direct evidence that Ca(2+) signaling can influence Zn(2+) homeostasis and vice versa, that Zn(2+) dynamics may modulate Ca(2+) signaling.

Cloning of casbene and neocembrene synthases from Euphorbiaceae plants and expression in Saccharomyces cerevisiae.

A large number of diterpenes have been isolated from Euphorbiaceae plants, many of which are of interest due to toxicity or potential therapeutic activity. Specific Euphorbiaceae diterpenes of medical interest include the latent HIV-1 activator prostratin (and related 12-deoxyphorbol esters), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. In spite of the large number of diterpenes isolated from these plants and the similarity of their core structures, there is little known about their biosynthetic pathways. Other than the enzymes involved in gibberellin biosynthesis, the only diterpene synthase isolated to date from the Euphorbiaceae has been casbene synthase, responsible for biosynthesis of a macrocyclic diterpene in the castor bean (Ricinus communis). Here, we have selected five Euphorbiaceae species in which to investigate terpene biosynthesis and report on the distribution of diterpene synthases within this family. We have discovered genes encoding putative casbene synthases in all of our selected Euphorbiaceae species and have demonstrated high-level casbene production through expression of four of these genes in a metabolically engineered strain of Saccharomyces cerevisiae. The only other diterpene synthase found among the five plants was a neocembrene synthase from R. communis (this being the first report of a neocembrene synthase gene). Based on the prevalence of casbene synthases, the lack of other candidates, and the structure of the casbene skeleton, we consider it likely that casbene is the precursor to a large number of Euphorbiaceae diterpenes. Casbene production levels of 31 mg/L were achieved in S. cerevisiae and we discuss strategies to further increase production by maximizing flux through the mevalonate pathway.

Focal adhesion disassembly is an essential early event in hepatic stellate cell chemotaxis.

Chemotaxis (i.e., directed migration) of hepatic stellate cells to areas of inflammation is a requisite event in the liver's response to injury. Previous studies of signaling pathways that regulate stellate cell migration suggest a key role for focal adhesions, but the exact function of these protein complexes in motility remains unclear. Focal adhesions attach a cell to its substrate and therefore must be regulated in a highly coordinated manner during migration. To test the hypothesis that focal adhesion turnover is an essential early event for chemotaxis in stellate cells, we employed a live-cell imaging technique in which chemotaxis was induced by locally stimulating the tips of rat stellate cell protrusions with platelet-derived growth factor-BB (PDGF). Focal adhesions were visualized with an antibody directed against vinculin, a structural component of the focal adhesion complex. PDGF triggered rapid disassembly of adhesions within 6.25 min, subsequent reassembly by 12.5 min, and continued adhesion assembly in concert with the spreading protrusion until the completion of chemotaxis. Blockade of adhesion disassembly by growing cells on fibronectin or treatment with nocodazole prevented a chemotactic response to PDGF. Augmentation of adhesion disassembly with ML-7 enhanced the chemotactic response to PDGF. These data suggest that focal adhesion disassembly is an essential early event in stellate cell chemotaxis in response to PDGF.