Biology subjects

Jiou, J.

Publications and source records attributed to Jiou, J..

7 recordsLinked to original sources

bioRxiv2025

Multi-immersion Oblique Plane Microscope (miOPM): A reconfigurable platform for high-resolution Light-Sheet Fluorescence Microscopy.

Light-Sheet Fluorescence Microscopy (LSFM) enables gentle, rapid, and efficient volumetric imaging of biological specimens. Despite its potential, LSFM has fragmented into multiple variants, each narrowly optimized for specific samples or imaging regimes, limiting its broad applicability. To overcome this barrier, we introduce the multi-immersion oblique plane microscope (miOPM), an adaptable LSFM platform for high-resolution imaging ranging from subcellular dynamics to whole organisms and cleared tissues. miOPM uniquely supports seamless interchangeability of oil, water, and air objectives, and maintains diffraction limited performance across a refractive index range of 1.33 - 1.51. It is compatible with standard sample mounting, current clearing protocols and high-throughput 3D imaging. We leverage miOPM to image diverse biological specimens at multiple spatial scales. By greatly expanding the adaptability and ease of use of LSFM, miOPM is poised to democratize advanced three-dimensional imaging.

bioengineering↗

bioRxiv2024

The RBR E3 ubiquitin ligase HOIL-1 can ubiquitinate diverse non-proteinaceous substrates in vitro

HOIL-1 is a RING-between-RING (RBR)-family E3 ubiquitin ligase and component of the linear ubiquitin chain assembly complex (LUBAC). While most E3 ubiquitin ligases conjugate ubiquitin to protein lysine sidechains, HOIL-1 has been reported to ubiquitinate hydroxyl groups in protein serine and threonine sidechains and glucosaccharides, such as glycogen and its building block maltose, in vitro. However, HOIL-1 substrate specificity is currently poorly defined. Here we show that HOIL-1 is unable to ubiquitinate lysine but can efficiently ubiquitinate serine as well as a variety of model and physiologically relevant di- and monosaccharides in vitro. We identify a critical catalytic histidine residue, His510, in the flexible catalytic site of HOIL-1 that enables this O-linked ubiquitination and prohibits ubiquitin discharge onto lysine sidechains. Finally, we utilise HOIL-1s in vitro non-proteinaceous ubiquitination activity and an engineered, constitutively active HOIL-1 variant to produce preparative amounts of different ubiquitinated saccharides that can be used as tool compounds and standards in the rapidly emerging field of non-proteinaceous ubiquitination.

biochemistry↗

bioRxiv2024

Phosphate-dependent nuclear export via a novel NES class recognized by exportin Msn5

Gene expression in response to environmental stimuli is dependent on nuclear localization of key signaling components, which can be tightly regulated by phosphorylation. This is exemplified by the phosphate-sensing transcription factor Pho4, which requires phosphorylation for nuclear export by the yeast exportin Msn5. Unlike the traditional hydrophobic nuclear export signal (NES) utilized by the Exportin-1/XPO1 system, cryogenic-electron microscopy structures reveal that Pho4 presents a novel, phosphorylated 35-residue NES that interacts with the concave surface of Msn5 through two Pho4 phospho-serines that align with two Msn5 basic patches, unveiling a previously unknown mechanism of phosphate-specific recognition. Furthermore, the discovery that unliganded Msn5 is autoinhibited explains the positive cooperativity of Pho4/Ran-binding and proposes a mechanism for Pho4s release in the cytoplasm. These findings advance our understanding of the diversity of signals that drive nuclear export and how cargo phosphorylation is crucial in regulating nuclear transport and controlling cellular signaling pathways.

biophysics↗

bioRxiv2023

Structural mechanism of co-import of H2A-H2B and its chaperone Nap1 by nuclear import receptor Kap114

Core histones are synthesized and processed in the cytoplasm before transport into the nucleus for assembly into nucleosomes; however, they must also be chaperoned as free histones are toxic. The importin Kap114 binds and transports histone H2A-H2B into the yeast nucleus, where RanGTP facilitates H2A-H2B release. Kap114 and H2A-H2B also bind the Nap1 histone chaperone, which is found in both the cytoplasm and the nucleus, but how Nap1 and Kap114 cooperate in H2A-H2B processing and nucleosome assembly has been unclear. To understand these mechanisms, we used biochemical and structural analyses to reveal how Nap1, Kap114, H2A-H2B and RanGTP interact. We show that Kap114, H2A-H2B and a Nap1 dimer (Nap12) assemble into a 1:1:1 ternary complex. Cryogenic electron microscopy revealed two distinct Kap114/Nap12/H2A-H2B structures: one of H2A-H2B sandwiched between Nap12 and Kap114, and another in which Nap12 bound to the Kap114*H2A-H2B complex without contacting H2A-H2B. Another Nap12*H2A-H2B*Kap114*RanGTP structure reveals the nuclear complex. Mutagenesis revealed shared critical interfaces in all three structures. Consistent with structural findings, DNA competition experiments demonstrated that Kap114 and Nap12 together chaperone H2A-H2B better than either protein alone. When RanGTP is present, Kap114s chaperoning activity diminishes. However, the presence of Nap12 within the Nap12*H2A-H2B*Kap114*RanGTP quaternary complex restores its ability to chaperone H2A-H2B. This complex effectively deposits H2A-H2B into nucleosomes. Together, these findings suggest that Kap114 and Nap12 provide a sheltered path from cytoplasm to nucleus, facilitating the transfer of H2A-H2B from Kap114 to Nap12, ultimately directing its specific deposition into nucleosomes. Significance StatementFree histones are toxic and must be sequestered by other macromolecules in the cell. Nuclear import receptor Kap114 imports H2A-H2B into the nucleus while also chaperoning it. The histone chaperone Nap1 also chaperones H2A-H2B, but it is unclear how Nap1 and Kap114 cooperate to process H2A-H2B. We present biochemical and structural results that explain how Kap114, Nap1 and H2A-H2B assemble in the absence and presence of RanGTP, how Nap1 and Kap114 co-chaperone H2A-H2B, and how RanGTP and Nap1 coordinate the transfer of H2A-H2B from Kap114 to assembling nucleosomes in the nucleus.

biophysics↗

bioRxiv2023

Molecular basis of RanGTP-activated nucleosome assembly with Histones H2A-H2B bound to Importin-9

Padavannil et al. 2019 show that Importin-9 (Imp9) transports Histones H2A-H2B from the cytoplasm to the nucleus using a non-canonical mechanism whereby binding of a GTP-bound Ran GTPase (RanGTP) fails to evict the H2A-H2B cargo. Instead, a stable complex forms, comprised of equimolar RanGTP, Imp9, and H2A-H2B. Unlike the binary Imp9*H2A-H2B complex, this RanGTP*Imp9*H2A-H2B ternary complex can release H2A-H2B to an assembling nucleosome. Here, we define the molecular basis for this RanGTP-activated nucleosome assembly by Imp9. We use hydrogen-deuterium exchange coupled with mass spectrometry and compare the dynamics and interfaces of the RanGTP*Imp9*H2A-H2B ternary complex to those in the Imp9*H2A-H2B or Imp9*RanGTP binary complexes. Our data are consistent with the Imp9*H2A-H2B structure by Padavannil et al. 2019 showing that Imp9 HEAT repeats 4-5 and 18-19 contact H2A-H2B, as well as many homologous importin*RanGTP structures showing that importin HEAT repeats 1 and 3, and the h8 loop, contact RanGTP. We show that Imp9 stabilizes H2A-H2B beyond the direct binding site, similar to other histone chaperones. Importantly, we reveal that binding of RanGTP releases H2A-H2B interaction at Imp9 HEAT repeats 4-5, but not 18-19. This exposes DNA- and histone-binding surfaces of H2A-H2B, thereby facilitating nucleosome assembly. We also reveal that RanGTP has a weaker affinity for Imp9 when H2A-H2B is bound. This may ensure that H2A-H2B is only released in high RanGTP concentrations near chromatin. We delineate the molecular link between the nuclear import of H2A-H2B and its deposition into chromatin by Imp9. SignificanceImp9 is the primary importin for shuttling H2A-H2B from the cytoplasm to the nucleus. It employs an unusual mechanism where the binding of RanGTP alone is insufficient to release H2A-H2B. The resulting stable RanGTP*Imp9*H2A-H2B complex gains nucleosome assembly activity as H2A-H2B can be deposited onto an assembling nucleosome. We show that H2A-H2B is allosterically stabilized via interactions with both N- and C-terminal portions of Imp9, reinforcing its chaperone-like behavior. RanGTP binding causes H2A-H2B release from the N-terminal portion of Imp9 only. The newly-exposed H2A-H2B surfaces can interact with DNA or H3-H4 in nucleosome assembly. Imp9 thus plays a multi-faceted role in histone import, storage, and deposition regulated by RanGTP, controlling histone supply in the nucleus and to chromatin.

biophysics↗

bioRxiv2022

Mechanism of RanGTP priming the release of H2A-H2B from Kap114 and Importin-9

Previously we showed that the nuclear import receptor Importin-9 wraps around the H2A-H2B core to chaperone and transport it from the cytoplasm to the nucleus. However, unlike most nuclear import systems where RanGTP dissociates cargoes from their importins, RanGTP binds stably to the Importin-9*H2A-H2B complex and formation of the ternary RanGTP*Importin-9*H2A-H2B complex facilitates H2A-H2B release to the assembling nucleosome. It was unclear how RanGTP and the cargo H2A-H2B can bind simultaneously to an importin, and how interactions of the three components position H2A-H2B for nucleosome assembly. Here we show cryo-EM structures of Importin-9*RanGTP and of its yeast homolog Kap114, including Kap114*RanGTP, Kap114*H2A-H2B, and RanGTP*Kap114*H2A-H2B to explain how the conserved Kap114 binds H2A-H2B and RanGTP simultaneously and how the GTPase primes histone transfer to the nucleosome. In the ternary complex, RanGTP binds to the N-terminal repeats of Kap114 in the same manner as in the Kap114/Importin-9*RanGTP complex, and H2A-H2B binds via its acidic patch to the Kap114 C-terminal repeats much like in the Kap114/Importin-9*H2A-H2B complex. Ran binds to a different conformation of Kap114 in the ternary RanGTP*Kap114*H2A-H2B complex. Here, Kap114 no longer contacts the H2A-H2B surface proximal to the H2A docking domain that drives nucleosome assembly, positioning it for transfer to the assembling nucleosome. Significance StatementHistones and their chaperone networks are typically conserved in eukaryotes. The yeast importin Kap114 and its human homolog Importin-9 share low sequence identity, but both are primary nuclear import receptors for the core histone heterodimer H2A-H2B. Cryo-EM structures of Kap114*H2A-H2B, Kap114*RanGTP and Importin-9*RanGTP complexes show homologous structure and function for Kap114 and Importin-9. In the nucleus, RanGTP binding to Kap114/Imp9*H2A-H2B does not release H2A-H2B, but RanGTP binds to form an atypical ternary complex. Structure of the ternary RanGTP*Kap114*H2A-H2B complex explains how the GTPase and cargo bind simultaneously to Kap114 and how the presence of Ran in the complex primes H2A-H2B transfer to assembling nucleosomes.

biochemistry↗

bioRxiv2018

Anatomy of a pressure sensing protein kinase

Cells respond to hydrostatic pressure to maintain cellular, organ, and organism level functions, yet the direct pressure sensors are largely unknown. Here we show that hydrostatic pressure directly activates With No Lysine(K) kinase-3 (WNK3) 1, a soluble intracellular protein kinase. Using gel filtration we demonstrate that pressure induces a dimer to monomer transition in a construct of the unphosphorylated kinase domain of WNK3 (uWNK3-KDm or uWNK3). The uWNK3 has not been crystallized, but crosslinking data suggest that the uWNK3 dimer corresponds to crystallographically observed dimer of WNK1 (uWNK1-KDm, or uWNK1) 2,3. Sequence alignments with WNKs from species living in different pressure environments and mutational analysis lend further support for this idea. Unique features of the uWNK1 structure suggest a mechanism involving bound water. We further show that hydrostatic pressure activates full-length WNK3 in D. melanogaster tubules.

biophysics↗