Quantum Dot-Based Three-Dimensional Single-Particle Tracking Characterizes the Evolution of Spatiotemporal Heterogeneity in Necrotic Cells.

Cell death is a fundamental biological process with different modes including apoptosis and necrosis. In contrast to programmed apoptosis, necrosis was previously considered disordered and passive, but it is now being realized to be under regulation by certain biological pathways. However, the intracellular dynamics that coordinates with cellular structure changes during necrosis remains unknown, limiting our understanding of the principles of necrosis. Here, we characterized the spatiotemporal intracellular diffusion dynamics in cells undergoing necrosis, using three-dimensional single-particle tracking of quantum dots. We found temporally increased diffusion rates in necrotic cells and spatially enhanced diffusion heterogeneity in the cell periphery, which could be attributed to the reduced molecular crowding resulting from cell swelling and peripheral blebbing, respectively. Moreover, the three-dimensional intracellular diffusion transits from strong anisotropy to nearly isotropy, suggesting a remodeling of the cytoarchitecture that relieves the axial constraint on intracellular diffusion during necrosis. Our results reveal the remarkable alterations of intracellular diffusion dynamics and biophysical properties in necrosis, providing insight into the well-organized nonequilibrium necrotic cell death from a biophysical perspective.

H2A mono-ubiquitination differentiates FACT's functions in nucleosome assembly and disassembly.

The histone chaperone FACT (FAcilitates Chromatin Transcription) plays an essential role in transcription and DNA replication by its dual functions on nucleosome assembly to maintain chromatin integrity and nucleosome disassembly to destabilize nucleosome and facilitate its accessibility simultaneously. Mono-ubiquitination at Lysine 119 of H2A (ubH2A) has been suggested to repress transcription by preventing the recruitment of FACT at early elongation process. However, up to date, how ubH2A directly affects FACT on nucleosome assembly and disassembly remains elusive. In this study, we demonstrated that the dual functions of FACT are differently regulated by ubH2A. The H2A ubiquitination does not affect FACT's chaperone function in nucleosome assembly and FACT can deposit ubH2A-H2B dimer on tetrasome to form intact nucleosome. However, ubH2A greatly restricts FACT binding on nucleosome and inhibits its activity of nucleosome disassembly. Interestingly, deubiquitination of ubH2A rescues the nucleosome disassembly function of FACT to activate gene transcription. Our findings provide mechanistic insights of how H2A ubiquitination affects FACT in breaking nucleosome and maintaining its integrity, which sheds light on the biological function of ubH2A and various FACT's activity under different chromatin states.

Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi‡.

Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features.

Structural and functional studies of SF1B Pif1 from Thermus oshimai reveal dimerization-induced helicase inhibition.

Pif1 is an SF1B helicase that is evolutionarily conserved from bacteria to humans and plays multiple roles in maintaining genome stability in both nucleus and mitochondria. Though highly conserved, Pif1 family harbors a large mechanistic diversity. Here, we report crystal structures of Thermus oshimai Pif1 (ToPif1) alone and complexed with partial duplex or single-stranded DNA. In the apo state and in complex with a partial duplex DNA, ToPif1 is monomeric with its domain 2B/loop3 adopting a closed and an open conformation, respectively. When complexed with a single-stranded DNA, ToPif1 forms a stable dimer with domain 2B/loop3 shifting to a more open conformation. Single-molecule and biochemical assays show that domain 2B/loop3 switches repetitively between the closed and open conformations when a ToPif1 monomer unwinds DNA and, in contrast with other typical dimeric SF1A helicases, dimerization has an inhibitory effect on its helicase activity. This mechanism is not general for all Pif1 helicases but illustrates the diversity of regulation mechanisms among different helicases. It also raises the possibility that although dimerization results in activation for SF1A helicases, it may lead to inhibition for some of the other uncharacterized SF1B helicases, an interesting subject warranting further studies.

Diffusion Behaviors of Integrins in Single Cells Altered by Epithelial to Mesenchymal Transition.

Cell morphology and migration depend critically on the adhesions on the extracellular matrix (ECM), determined by the transmembrane protein integrins. The epithelial to mesenchymal transition (EMT) is a prominent transformation process in which adherent cells acquire a mesenchymal phenotype and a promoted migration. EMT plays important roles in embryonic development and cancer metastasis, and its hallmarks include the acquisition of front-back cell polarity and loss of cell-cell contact. However, how integrins dynamically regulate cell-ECM adhesions and cellular behaviors during EMT is still unclear. Using single-particle tracking of ß1-integrins labeled with quantum dots, the temporal-spatial on-membrane dynamics of integrins in the EMT of MCF10A cells is revealed. ß1-integrins exhibit significantly enhanced dynamics, which temporally behave more diffusive and less immobilized, and spatially become distributed asymmetrically with front regions being more dynamic. These dynamic alterations are shown to arise from microtubule remodeling in EMT. The results shed new light on the EMT mechanism from the cell-ECM adhesion perspective, and suggest that the enhanced integrin diffusion may represent as a new hallmark of EMT.

DNA polymerase Gp90 activities and regulations on strand displacement DNA synthesis revealed at single-molecule level.

Strand displacement DNA synthesis (SDDS) is an essential step in DNA replication. With magnetic tweezers, we investigated SDDS kinetics of wild-type gp90 and its exonuclease-deficient polymerase gp90 exo- at single-molecule level. A novel binding state of gp90 to the fork flap was confirmed prior to SDDS, suggesting an intermediate in the initiation of SDDS. The rate and processivity of SDDS by gp90 exo- or wt-gp90 are increased with force and dNTP concentration. The rate and processivity of exonuclease by wt-gp90 are decreased with force. High GC content decreases SDDS and exonuclease processivity but increases exonuclease rate for wt-gp90. The high force and dNTP concentration and low GC content facilitate the successive SDDS but retard the successive exonuclease for wt-gp90. Furthermore, increasing GC content accelerates the transition from SDDS or exonuclease to exonuclease. This work reveals the kinetics of SDDS in detail and offers a broader cognition on the regulation of various factors on SDDS at single-polymerase level.

Curaxin-Induced DNA Topology Alterations Trigger the Distinct Binding Response of CTCF and FACT at the Single-Molecule Level.

The candidate anticancer drug curaxins can insert into DNA base pairs and efficiently inhibit the growth of various cancers. However, how curaxins alter the genomic DNA structure and affect the DNA binding property of key proteins remains to be clarified. Here, we first showed that curaxin CBL0137 strongly stabilizes the interaction between the double strands of DNA and reduces DNA bending and twist rigidity simultaneously, by single-molecule magnetic tweezers. More importantly, we found that CBL0137 greatly impairs the binding of CTCF but facilitates trapping FACT on DNA. We revealed that CBL0137 clamps the DNA double helix that may induce a huge barrier for DNA unzipping during replication and transcription and causes the distinct binding response of CTCF and FACT on DNA. Our work provides a novel mechanical insight into CBL0137's anticancer mechanisms at the nucleic acid level.

The HRDC domain oppositely modulates the unwinding activity of E. coli RecQ helicase on duplex DNA and G-quadruplex.

RecQ family helicases are highly conserved from bacteria to humans and have essential roles in maintaining genome stability. Mutations in three human RecQ helicases cause severe diseases with the main features of premature aging and cancer predisposition. Most RecQ helicases shared a conserved domain arrangement which comprises a helicase core, an RecQ C-terminal domain, and an auxiliary element helicase and RNaseD C-terminal (HRDC) domain, the functions of which are poorly understood. In this study, we systematically characterized the roles of the HRDC domain in E. coli RecQ in various DNA transactions by single-molecule FRET. We found that RecQ repetitively unwinds the 3'-partial duplex and fork DNA with a moderate processivity and periodically patrols on the ssDNA in the 5'-partial duplex by translocation. The HRDC domain significantly suppresses RecQ activities in the above transactions. In sharp contrast, the HRDC domain is essential for the deep and long-time unfolding of the G4 DNA structure by RecQ. Based on the observations that the HRDC domain dynamically switches between RecA core- and ssDNA-binding modes after RecQ association with DNA, we proposed a model to explain the modulation mechanism of the HRDC domain. Our findings not only provide new insights into the activities of RecQ on different substrates but also highlight the novel functions of the HRDC domain in DNA metabolisms.

Intracellular transport is accelerated in early apoptotic cells.

Intracellular transport of cellular proteins and organelles is critical for establishing and maintaining intracellular organization and cell physiology. Apoptosis is a process of programmed cell death with dramatic changes in cell morphology and organization, during which signaling molecules are transported between different organelles within the cells. However, how the intracellular transport changes in cells undergoing apoptosis remains unknown. Here, we study the dynamics of intracellular transport by using the single-particle tracking method and find that both directed and diffusive motions of endocytic vesicles are accelerated in early apoptotic cells. With careful elimination of other factors involved in the intracellular transport, the reason for the acceleration is attributed to the elevation of adenosine triphosphate (ATP) concentration. More importantly, we show that the accelerated intracellular transport is critical for apoptosis, and apoptosis is delayed when the dynamics of intracellular transport is regulated back to the normal level. Our results demonstrate the important role of transport dynamics in apoptosis and shed light on the apoptosis mechanism from a physical perspective.

Structural analysis reveals a "molecular calipers" mechanism for a LATERAL ORGAN BOUNDARIES DOMAIN transcription factor protein from wheat.

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) proteins, a family of plant-specific transcription factors harboring a conserved Lateral Organ Boundaries (LOB) domain, are regulators of plant organ development. Recent studies have unraveled additional pivotal roles of the LBD protein family beyond defining lateral organ boundaries, such as pollen development and nitrogen metabolism. The structural basis for the molecular network of LBD-dependent processes remains to be deciphered. Here, we solved the first structure of the homodimeric LOB domain of Ramosa2 from wheat (TtRa2LD) to 1.9 Å resolution. Our crystal structure reveals structural features shared with other zinc-finger transcriptional factors, as well as some features unique to LBD proteins. Formation of the TtRa2LD homodimer relied on hydrophobic interactions of its coiled-coil motifs. Several specific motifs/domains of the LBD protein were also involved in maintaining its overall conformation. The intricate assembly within and between the monomers determined the precise spatial configuration of the two zinc fingers that recognize palindromic DNA sequences. Biochemical, molecular modeling, and small-angle X-ray scattering experiments indicated that dimerization is important for cooperative DNA binding and discrimination of palindromic DNA through a molecular calipers mechanism. Along with previously published data, this study enables us to establish an atomic-scale mechanistic model for LBD proteins as transcriptional regulators in plants.