Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription.

In eukaryotes, RNA polymerase II (RNApII) transcribes messenger RNA from template DNA. Decades of experiments have identified the proteins needed for transcription activation, initiation complex assembly, and productive elongation. However, the dynamics of recruitment of these proteins to transcription complexes, and of the transitions between these steps, are poorly understood. We used multiwavelength single-molecule fluorescence microscopy to directly image and quantitate these dynamics in a budding yeast nuclear extract that reconstitutes activator-dependent transcription in vitro. A strong activator (Gal4-VP16) greatly stimulated reversible binding of individual RNApII molecules to template DNA. Binding of labeled elongation factor Spt4/5 to DNA typically followed RNApII binding, was NTP dependent, and was correlated with association of mRNA binding protein Hek2, demonstrating specificity of Spt4/5 binding to elongation complexes. Quantitative kinetic modeling shows that only a fraction of RNApII binding events are productive and implies a rate-limiting step, probably associated with recruitment of general transcription factors, needed to assemble a transcription-competent preinitiation complex at the promoter. Spt4/5 association with transcription complexes was slowly reversible, with DNA-bound RNApII molecules sometimes binding and releasing Spt4/5 multiple times. The average Spt4/5 residence time was of similar magnitude to the time required to transcribe an average length yeast gene. These dynamics suggest that a single Spt4/5 molecule remains associated during a typical transcription event, yet can dissociate from RNApII to allow disassembly of abnormally long-lived (i.e., stalled) elongation complexes.

Three dimensional evaluation of cerebrovascular density and branching in chronic traumatic encephalopathy.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease associated with exposure to repetitive head impacts (RHI) and characterized by perivascular accumulations of hyperphosphorylated tau protein (p-tau) at the depths of the cortical sulci. Studies of living athletes exposed to RHI, including concussive and nonconcussive impacts, have shown increased blood-brain barrier permeability, reduced cerebral blood flow, and alterations in vasoreactivity. Blood-brain barrier abnormalities have also been reported in individuals neuropathologically diagnosed with CTE. To further investigate the three-dimensional microvascular changes in individuals diagnosed with CTE and controls, we used SHIELD tissue processing and passive delipidation to optically clear and label blocks of postmortem human dorsolateral frontal cortex. We used fluorescent confocal microscopy to quantitate vascular branch density and fraction volume. We compared the findings in 41 male brain donors, age at death 31-89 years, mean age 64 years, including 12 donors with low CTE (McKee stage I-II), 13 with high CTE (McKee stage III-IV) to 16 age- and sex-matched non-CTE controls (7 with RHI exposure and 9 with no RHI exposure). The density of vessel branches in the gray matter sulcus was significantly greater in CTE cases than in controls. The ratios of sulcus versus gyrus vessel branch density and fraction volume were also greater in CTE than in controls and significantly above one for the CTE group. Hyperphosphorylated tau pathology density correlated with gray matter sulcus fraction volume. These findings point towards increased vascular coverage and branching in the dorsolateral frontal cortex (DLF) sulci in CTE, that correlates with p-tau pathology.

Volumetric Characterization of Microvasculature in Ex Vivo Human Brain Samples By Serial Sectioning Optical Coherence Tomography.

OBJECTIVE: Serial sectioning optical coherence tomography (OCT) enables accurate volumetric reconstruction of several cubic centimeters of human brain samples. We aimed to identify anatomical features of the ex vivo human brain, such as intraparenchymal blood vessels and axonal fiber bundles, from the OCT data in 3D, using intrinsic optical contrast. METHODS: We developed an automatic processing pipeline to enable characterization of the intraparenchymal microvascular network in human brain samples. RESULTS: We demonstrated the automatic extraction of the vessels down to a 20 µm in diameter using a filtering strategy followed by a graphing representation and characterization of the geometrical properties of microvascular network in 3D. We also showed the ability to extend this processing strategy to extract axonal fiber bundles from the volumetric OCT image. CONCLUSION: This method provides a viable tool for quantitative characterization of volumetric microvascular network as well as the axonal bundle properties in normal and pathological tissues of the ex vivo human brain.

Apical pollen tube wall curvature correlates with growth and indicates localized changes in the yielding of the cell wall.

The shape of the apical region of lily pollen tube changes rhythmically as the growth rate of the tube oscillates becoming alternately more prolate then back to oblate. We quantified shape change by calculating the curvature of the cross-sectional edge of the pollen tube tip and cross-correlating curvature changes with growth rate. The apical region takes the form of a partial elliptical spheroid, with variation in the length and location of the minor axis. During oscillation curvature profiles show a sharp increase in curvature at the "shoulders" of the apex when oblate, 4-7 µm from the flatter central zone. As the tip becomes more prolate, the "shoulders" decrease rapidly in curvature and move towards the growth axis as curvature at the tip increases. We understand curvature changes to represent differential changes in local wall expansion rates, driven by uniform turgor pressure and mediated by changes in wall polysaccharides. To become more oblate, the tip region must become less extensible than the "shoulder" region. And, as the tip becomes more prolate, the increased curvature must be due to increased local expansion. We found that changes in the growth velocity of the "shoulders" of the cell measured as the progress of the cell edge along the growth axis are cyclically out of phase with growth velocity at the tip such that the shoulder regions lag for part of the oscillation cycle, then "catch up" as the growth rate at the tip reaches a maximum and begins to decline. In this way the cell becomes oblate. Cell shape and growth rate oscillate in concert and are functionally related. Spatial change in edge growth rate points to important cellular locations for further investigation of vesicle movement and exocytosis, calcium gradients, and actin dynamics in lily pollen tubes.