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Bioprinting allows the manufacture of complex cell-laden hydrogel constructs that can mature into tissue replacements in subsequent cell culture processes. The nozzles used in currently available bioprinters limit the print resolution and at dimensions below 100 µm clogging is expected. Most critically, the reduction of nozzle diameter also increases shear stress during printing. At critical shear stress, mechanical damage to printed cells triggers cell death. To overcome these limitations, a novel 3D bioprinting method based on the principle of acoustic droplet ejection (ADE) is introduced here. The absence of a nozzle in this method minimizes critical shear stress. A numerical simulation reveals that maximum shear stress during the ADE process is 2.7 times lower than with a Ø150 µm microvalve nozzle. Printing of cell clusters contained in droplets at the millimeter length scale, as well as in droplets the size of a single cell, is feasible. The precise 3D build-up of cell-laden structures is demonstrated and evidence is provided that there are no negative effects on stem cell morphology, proliferation, or differentiation capacities. This multiscale acoustic bioprinting technique thus holds promise for cell-preserving creation of complex and individualized cell-laden 3D hydrogel structures.

Two ubiquitin-conjugating enzymes, RAD6 and the heteromeric UBC13-MMS2 complex, have been implicated in post-replicative DNA damage repair in yeast. Here we provide a mechanistic basis for cooperation between the two enzymes. We show that two chromatin-associated RING finger proteins, RAD18 and RAD5, play a central role in mediating physical contacts between the members of the RAD6 pathway. RAD5 recruits the UBC13-MMS2 complex to DNA by means of its RING finger domain. Moreover, RAD5 association with RAD18 brings UBC13-MMS2 into contact with the RAD6-RAD18 complex. Interaction between the two RING finger proteins thus promotes the formation of a heteromeric complex in which the two distinct ubiquitin-conjugating activities of RAD6 and UBC13-MMS2 can be closely coordinated. Surprisingly, UBC13 and MMS2 are largely cytosolic proteins, but DNA damage triggers their redistribution to the nucleus. These findings suggest a mechanism by which the activity of this DNA repair pathway could be regulated.

Selective degradation of proteins at the endoplasmic reticulum (ER-associated degradation) is thought to proceed largely via the cytosolic ubiquitin-proteasome pathway. Recent data have indicated that the dislocation of short-lived integral-membrane proteins to the cytosolic proteolytic system may require components of the Sec61 translocon. Here we show that the proteasome itself can participate in the extraction of an ER-membrane protein from the lipid bilayer. In yeast mutants expressing functionally attenuated proteasomes, degradation of a short-lived doubly membrane-spanning protein proceeds rapidly through the N-terminal cytosolic domain of the substrate, but slows down considerably when continued degradation of the molecule requires membrane extraction. Thus, proteasomes engaged in ER degradation can directly process transmembrane proteins through a mechanism in which the dislocation of the substrate and its proteolysis are coupled. We therefore propose that the retrograde transport of short-lived substrates may be driven through the activity of the proteasome.

The related transcription factors ACE1 of Saccharomyces cerevisiae and AMT1 of Candida glabrata are involved in copper metabolism by activating the transcription of copper metallothionein genes. ACE1 and AMT1 are 'copper-fist' transcription factors which possess a conserved cysteine-rich copper binding domain required for DNA binding. Here we report the identification of a nuclear protein from S. cerevisiae, MAC1, whose N-terminal region is highly similar to the copper and DNA binding domains of ACE1 and AMT1. Loss-of-function mutants of MAC1 have a defect in the plasma membrane Cu(II) and Fe(III) reductase activity, are slow growing, respiratory deficient, and hypersensitive to heat and exposure to cadmium, zinc, lead and H2O2. Conversely, a dominant gain-of-function mutant of MAC1 shows an elevated reductase activity and is hypersensitive to copper. We have identified two target genes of MAC1 whose altered expression in mutants of MAC1 can account for some of the observed mutant phenotypes. First, MAC1 is involved in basal level transcription of FRE1, encoding a plasma membrane component associated with both Cu(II) and Fe(III) reduction. Second, MAC1 is involved in the H2O2-induced transcription of CTT1, encoding the cytosolic catalase. This suggests that MAC1 may encode a novel metal-fist transcription factor required for both basal and regulated transcription of genes involved in Cu/Fe utilization and the stress response.

The covalent attachment of ubiquitin to cellular proteins is catalyzed by members of a family of ubiquitin-conjugating enzymes. These enzymes participate in a variety of cellular processes, including selective protein degradation, DNA repair, cell cycle control, and sporulation. In the yeast Saccharomyces cerevisiae, two closely related ubiquitin-conjugating enzymes, UBC4 and UBC5, have recently been shown to mediate the selective degradation of short-lived and abnormal proteins. We have now identified a third distinct member of this class of ubiquitin-conjugating enzymes, UBC1. UBC1, UBC4 and UBC5 are functionally overlapping and constitute an enzyme family essential for cell growth and viability. All three mediate selective protein degradation, however, UBC1 appears to function primarily in the early stages of growth after germination of spores. ubc1 mutants generated by gene disruption display only a moderate slow growth phenotype, but are markedly impaired in growth following germination. Moreover, yeast carrying the ubc1ubc4 double mutation are viable as mitotic cells, however, these cells fail to survive after undergoing sporulation and germination. This specific requirement for UBC1 after a state of quiescence suggests that degradation of certain proteins may be crucial at this transition point in the yeast life cycle.

Ubiquitin-conjugating enzymes catalyse the covalent attachment of ubiquitin to target proteins. Members of this enzyme family are involved in strikingly diverse cellular functions: UBC2 (RAD6) is central to DNA repair, UBC3 (CDC34) is involved in cell cycle control. We have cloned the genes for two novel ubiquitin-conjugating enzymes, UBC4 and UBC5, from the yeast Saccharomyces cerevisiae. These enzymes mediate selective degradation of short-lived and abnormal proteins. UBC4 and UBC5 are closely related in sequence and complementing in function. Expression of UBC4 and UBC5 genes is heat inducible. UBC4 and UBC5 enzymes generate high mol. wt ubiquitin-protein conjugates in vivo consistent with previous studies which suggested that attachment of multiple ubiquitin molecules to proteolytic substrates is required for their selective degradation. UBC4 and UBC5 enzymes comprise a major part of total ubiquitin-conjugation activity in stressed cells. Turnover of short-lived proteins and canavanyl-peptides but not of long-lived proteins is markedly reduced in ubc4ubc5 mutants. Loss of UBC4 and UBC5 activity impairs cell growth, leads to inviability at elevated temperatures or in the presence of an amino acid analog, and induces the stress response.