Cytoplasmic division cycles without the nucleus and mitotic CDK/cyclin complexes.

Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. We demonstrate in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, we present evidence that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. We postulate that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes.

Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers.

Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remain unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1's lumenal domain. Phosphorylation of IRE1's kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans-autophosphorylation, which in turn drives IRE1's RNase activity.

Our cells contain many different compartments that each perform specific tasks. A cellular compartment known as the endoplasmic reticulum is responsible for making many of the proteins the cell requires and transporting them around the cell. It is important that the endoplasmic reticulum remains healthy and, therefore, cells use a protein called IRE1 that senses when this compartment is under stress. IRE1 then sends a signal to the control center of the cell (known as the nucleus) to ask for help. Previous studies suggest that IRE1 assembles into either pairs or larger groups of molecules known as oligomers when it senses that the endoplasmic reticulum is under stress. However, it remains unclear whether such assembly is the main switch that turns IRE1 on and, if so, how many molecules need to come together to flip the switch. Here, Belyy et al. genetically engineered human bone cancer cells to attach a mark known as a HaloTag to IRE1.The team developed a microscopy approach to count, in living cells, how many tagged IRE1 molecules join. The experiments indicated that IRE1 proteins were generally found as pairs in unstressed cells. When the endoplasmic reticulum experienced stress, IRE1 proteins briefly assembled into oligomers before disassembling back into pairs. Mutated versions of IRE1 revealed the exact parts of IRE1 that connect the pairs and the larger oligomers. These findings suggest that the assembly of IRE1 pairs into oligomers plays a major part in the activation of IRE1 to send a stress signal to the nucleus. IRE1 signaling is closely implicated in both cancer biology and aging, and therefore, understanding how it works may aid the development of new therapies for cancer, dementia, and other health conditions affecting older people. Furthermore, the microscopy approach developed in this work could be adapted to study other proteins that relay signals in living cells.

Autonomous clocks that regulate organelle biogenesis, cytoskeletal organization, and intracellular dynamics.

How do cells perceive time? Do cells use temporal information to regulate the production/degradation of their enzymes, membranes, and organelles? Does controlling biological time influence cytoskeletal organization and cellular architecture in ways that confer evolutionary and physiological advantages? Potential answers to these fundamental questions of cell biology have historically revolved around the discussion of 'master' temporal programs, such as the principal cyclin-dependent kinase/cyclin cell division oscillator and the circadian clock. In this review, we provide an overview of the recent evidence supporting an emerging concept of 'autonomous clocks,' which under normal conditions can be entrained by the cell cycle and/or the circadian clock to run at their pace, but can also run independently to serve their functions if/when these major temporal programs are halted/abrupted. We begin the discussion by introducing recent developments in the study of such clocks and their roles at different scales and complexities. We then use current advances to elucidate the logic and molecular architecture of temporal networks that comprise autonomous clocks, providing important clues as to how these clocks may have evolved to run independently and, sometimes at the cost of redundancy, have strongly coupled to run under the full command of the cell cycle and/or the circadian clock. Next, we review a list of important recent findings that have shed new light onto potential hallmarks of autonomous clocks, suggestive of prospective theoretical and experimental approaches to further accelerate their discovery. Finally, we discuss their roles in health and disease, as well as possible therapeutic opportunities that targeting the autonomous clocks may offer.