During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round. Mitotic cell rounding is thought to facilitate organization within the mitotic cell and be necessary for the geometric requirements of division. However, the forces that drive this shape change remain poorly understood in the presence of external impediments, such as a tissue environment. Here we use cantilevers to track cell rounding force and volume. We show that cells have an outward rounding force, which increases as cells enter mitosis. We find that this mitotic rounding force depends both on the actomyosin cytoskeleton and the cells' ability to regulate osmolarity. The rounding force itself is generated by an osmotic pressure. However, the actomyosin cortex is required to maintain this rounding force against external impediments. Instantaneous disruption of the actomyosin cortex leads to volume increase, and stimulation of actomyosin contraction leads to volume decrease. These results show that in cells, osmotic pressure is balanced by inwardly directed actomyosin cortex contraction. Thus, by locally modulating actomyosin-cortex-dependent surface tension and globally regulating osmotic pressure, cells can control their volume, shape and mechanical properties.

Actomyosin-dependent mitotic rounding occurs in both cell culture and tissue, where it is involved in cell positioning and epithelial organization. How actomyosin is regulated to mediate mitotic rounding is not well understood. Here we characterize the mechanics of single mitotic cells while imaging actomyosin recruitment to the cell cortex. At mitotic onset, the assembly of a uniform DIAPH1-dependent F-actin cortex coincides with initial rounding. Thereafter, cortical enrichment of F-actin remains stable while myosin II progressively accumulates at the cortex, and the amount of myosin at the cortex correlates with intracellular pressure. Whereas F-actin provides only short-term (<10 s) resistance to mechanical deformation, myosin sustains intracellular pressure for a longer duration (>60 s). Our data suggest that progressive accumulation of myosin II to the mitotic cell cortex probably requires the Cdk1 activation of both p21-activated kinases, which inhibit myosin recruitment, and of Rho kinase, which stimulates myosin recruitment to the cortex.

To cleave DNA, Type III restriction enzymes must communicate the relative orientation of two asymmetric recognition sites over hundreds of base pairs. The basis of this long-distance communication, for which ATP hydrolysis by their helicase domains is required, is poorly understood. Several conflicting DNA-looping mechanisms have been proposed, driven either by active DNA translocation or passive 3D diffusion. Using single-molecule DNA stretching in combination with bulk-solution assays, we provide evidence that looping is both highly unlikely and unnecessary, and that communication is strictly confined to a 1D route. Integrating our results with previous data, a simple communication scheme is concluded based on 1D diffusion along DNA.T he ability of enzymes bound at distant DNA sites to communicate with each other via long-range interactions is an important biological theme. Very often the underlying genetic processes, such as gene silencing, site-specific recombination, restriction, etc., rely on energy-independent DNA looping (1). For many other processes, such as in mismatch repair (2) and for both Type I and III restriction enzymes (REs) (3, 4), the long-range interaction relies on ATP hydrolysis and in these cases the contribution of general passive three-dimensional (3D) looping to communication remains controversial.Restriction enzymes are a model family for studying longrange communication because the majority (and in particular all Type I and III REs) need to interact with two separate DNA sequences before cutting DNA. For the Type II REs, there is growing evidence that passive 3D DNA looping (Fig. 1A) is frequently used (5). In contrast, the Type I and III REs contain protein domains that are classified as Superfamily 2 (SF2) DNA helicases (6), and these domains are required for ATPdependent DNA communication (7,8). The role of the helicase domains in the Type I REs has been clearly defined ( Fig. 1 A); communication involves DNA loop extrusion driven by directional dsDNA translocation (9, 10), without DNA unwinding (11), with the motor making steps along the DNA of Ͻ2 bp and consuming on average one ATP for each bp moved (12). Cleavage occurs upon collision with a second translocating motor at random positions distant from the binding site (3). This is therefore a pure 1D directional communication process. In comparison, the communication mechanism for Type III REs has not been accurately defined and conflicting models have been proposed (4,13,14).Type III REs require two copies of their asymmetric recognition site in an indirectly repeated, head-to-head (HtH) orientation (15) (Fig. 1 A) cleaving the DNA 25-27 bp downstream of only one of the two sites. Given that Type III REs also require the ATPase activity of their SF2 helicase domains [albeit unrelated to Type I REs (6)], a Type I-like DNA loop translocation model was proposed in which translocation was unidirectional, accounting for the site-orientation preference (4). In support of this model, apparent DNA looping activity has been observed in ...

Cleavage of viral DNA by the bacterial Type III RestrictionModification enzymes requires the ATP-dependent long-range communication between a distant pair of DNA recognition sequences. The classical view is that Type III endonuclease activity is only activated by a pair of asymmetric sites in a specific head-to-head inverted repeat. Based on this assumption and due to the presence of helicase domains in Type III enzymes, various motor-driven DNA translocation models for communication have been suggested. Using both single-molecule and ensemble assays we demonstrate that Type III enzymes can also cleave DNA with sites in tail-to-tail repeat with high efficiency. The ability to distinguish both inverted repeat substrates from direct repeat substrates in a manner independent of DNA topology or accessory proteins can only be reconciled with an alternative sliding mode of communication.diffusion | helicase | motor | switch A lmost every genetic process requires protein complexes to interact simultaneously with specific DNA sites or structures that are located at-a-distance along the genome, including events in DNA replication, repair, recombination, and transcription. In many cases these so-called "long-range communications" are independent of the relative orientation of the interacting sites on the DNA (1). E.g., gene activation by remote enhancer elements is associated with random DNA looping between regulatory elements, i.e., chromatin loop formation (2). Such reactions can occur on DNA of any topology, and can even occur between sites on separate DNA molecules providing the local concentration is sufficiently elevated (1). In other cases however, a successful interaction only occurs when the sites are located on the same DNA in a specific relative orientation. The reaction can then be said to have "site-orientation selectivity," which can be achieved using both NTP-independent or NTP-dependent pathways:Site-specific recombinases (SSRs) have provided a mechanistic framework for NTP-independent communication (3-5). For example, the transposon-encoded resolvases have a strong preference to recombine sites in direct repeat on the same DNA (6). Site-orientation selectivity is important as uncontrolled rearrangements of DNA sequences may result in loss of function. For all SSRs studied to-date, the long-range communication occurs by the sites interacting via thermally driven three-dimensional diffusion, i.e., DNA looping (7). However, to achieve site-orientation selectivity, the geometry of the resulting site-site synapse is biased by an energetic "filter" that can be a preference for DNA substrates with a particular topology (e.g., 8) or the requirement for accessory DNA-binding factors (e.g., 9). For example, recombination by the resolvases requires the capture of three DNA nodes which are significantly favored by negative supercoiling (6,8,(10)(11)(12).For many processes that are NTP-dependent, site-orientation selectivity comes from directional one-dimensional motion along DNA. A classical example is transcription-...