20 research outputs found
Inscuteable regulates the Pins-Mud spindle orientation pathway.
No full textDuring asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). Here we investigate the mechanism of spindle orientation by the Insc-Pins complex. Previously, we defined two Pins spindle orientation pathways: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. In the current study, we have examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. We find that the Insc-Pins complex requires Gαi for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. Our results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud)
Inscuteable competes with Mud, but not Dlg, for binding to Pins.
No full text<p>a, GST-pulldowns of Inscuteable with different Pins constructs reveals that Inscuteable binds specifically to constructs containing the full array of Pins TPRs. b, Coimmunoprecipitations of endogenous proteins from wild-type L3 brain extracts demonstrate that Inscuteable and Mud form exclusive complexes with Pins. c, GST-pulldown using GST-Pins TPRs incubated with a constant amount of Mud and increasing MBP-Insc reveals effective competition between Mud & Insc for binding to Pins. d, A control titrations of MBP alone do not result in dissociation of Mud from GST-Pins. e, GST-pulldown using GST-Mud incubated with constant 2 uM Pins TPR and increasing amounts of MBP-Insc results in an approximately 1∶1 stoichiometric dissociation of Pins TPRs from GST-Mud. f, A control titration of MBP alone does not result in disruption of Pins-Mud binding. g, Fluorescence anisotropy of TMR-Mud with increasing amounts of Pins TPRs exhibits a robust association profile. h, Addition of Inscuteable to a pre-formed complex of 100 nM TMR-Mud & 1 uM Pins causes a dissociation of the Mud-Pins complex & reduction of TMR-Mud anisotropy. i, GST-pulldown using <i>in vitro</i> Aurora-A phosphorylated Pins 1–466 results in complex formation with the Dlg GK domain. AurA treatment of a pre-formed MBP-Insc/Pins 1–466 complex likewise is able to form a complex with the Dlg GK domain.</p
Inscuteable-mediated orientation of the mitotic spindle requires Gαi.
No full text<p>a, current model of Inscuteable function. Insc serves as a link between the apical PAR complex and the spindle-orienting Pins-Gái complex. b, Ed-Insc (green) transfected S2 cells randomly orient the mitotic spindle (red) with respect to the region that is enriched in Ed. Spindle alignment is measured by drawing a vector from the center of the crescent (arrow) to the center of the mitotic spindle and then along the axis (dashes). c, Expression of Gái (blue) with Ed-Insc (green)is able to confer moderate spindle orienting activity. d, Cumulative percentage plot of spindle angles measured in the S2 Echinoid induced-polarity assay for Ed-Insc and Ed-Insc+Gái compared to previously-published data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029611#pone.0029611-Johnston1" target="_blank">[17]</a>. In these plots, the cumulative percentage of cells with a spindle angle below a particular value (x-axis) is shown. High spindle orienting activity corresponds to a deflection to lower spindle angles whereas no activity is a line across the diagonal. e, Ed-Insc expression in S2 cells is sufficient to robustly recruit endogenous Pins from the cytoplasm to the region of Ed enrichment. f, Ed alone is unable to polarize endogenous Pins. e, g, Ed-Insc induces colocalization of endogenous Pins with overexpressed Gai. h, Ed-Pins is able to recruit endogenous Dlg. i, Co-expression of Gαi with Ed-Pins results in robust recruitment of endogenous Mud. j,k Ed-GFP is unable to recruit endogenous Dlg or Mud to the induced-polarity cortical domains. l, Ed-Insc is able to recruit Dlg to the cortex, similar to cells expressing Ed-GFP-Pins. m, Ed-Insc is not able to recruit Mud (red) to the Ed-crescent, even in the presence of Gái. Scale bars for all panels represent 5 µm.</p
Proposed model for Inscuteable regulation of spindle orientation.
No full text<p>a, In early interphase, Inscuteable recruits cortical Gαi-Pins to the apical cortex. Insc-bound Pins can scaffold for Dlg, allowing for early microtubule attachment, but inhibits binding of Mud, preventing ectopic microtubule shortening. b, after nuclear envelope breakdown and trafficking along the mitotic spindle, Mud from astral microtubules competes Pins away from Insc and allows for microtubule shortening.</p
Expression of Inscuteable in cells expressing constitutively-active Pins reduces spindle orientation to Dlg-like levels.
No full text<p>a, Co-expression of Ed-Pins 1–466 (green), which robustly orients the mitotic spindle, with Inscuteable (inset), reduces the levels of spindle orientation in adherent, polarized S2 cells. b, Cumulative percentage plot of spindle angles measured in cells co-expressing Ed-Pins 1–466 and Inscuteable compared to published data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029611#pone.0029611-Johnston1" target="_blank">[17]</a>.</p
Ultrasensitive Synthetic Protein Regulatory Networks Using Mixed Decoys
No full textCellular protein interaction networks exhibit sigmoidal
input–output
relationships with thresholds and steep responses (i.e., ultrasensitivity).
Although cooperativity can be a source of ultrasensitivity, we examined
whether the presence of “decoy” binding sites that are
not coupled to activation could also lead to this effect. To systematically
vary key parameters of the system, we designed a synthetic regulatory
system consisting of an autoinhibited PDZ domain coupled to an activating
SH3 domain binding site. In the absence of a decoy binding site, this
system is non-ultrasensitive, as predicted by modeling of this system.
Addition of a high-affinity decoy site adds a threshold, but the response
is not ultrasensitive. We found that sigmoidal activation profiles
can be generated utilizing multiple decoys with mixtures of high and
low affinities, where high affinity decoys act to set the threshold
and low affinity decoys ensure a sigmoidal response. Placing the synthetic
decoy system in a mitotic spindle orientation cell culture system
thresholds this physiological activity. Thus, simple combinations
of non-activating binding sites can lead to complex regulatory responses
in protein interaction networks
Musikstädte as real and imaginary soundscapes: urban musical images as literary motifs in twentieth-century German modernism
No full textPhDThis study examines German literary images of musical life as part of the wider sound identity of the modern German city at the turn of the twentieth century. Focussing on a forty-year period from 1890 to 1930, synonymous with the emergence of the modern German metropolis as an aesthetic object, the project assesses, compares and contrasts how musical life in the Musikstädte was perceived and portrayed by writers in an increasingly noisy urban environment. How does urban musical life influence and condition city writings? What are the differences and similarities between the writings on various musical cities? Can an urban textual sound identity be derived from these differences and similarities? The approach employed to answer these questions is a new, cross-disciplinary one to urban sound in literature, moving beyond reading the key sounds of the urban soundscape using urban musicology, sensorial anthropology and cultural poetics towards a literary contextualisation of the urban aural experience.
The literary motifs of the symphony, the gramophone and urban noise are put under the spotlight through the analysis of a wide range of modernist works by authors who have a special relationship with music. At the centre of this analysis are the Kaffeehausliteratur authors Hermann Bahr, Alfred Polgar and Peter Altenberg, the then Munich-based author Thomas Mann and the lesser known René Schickele. The analysis of these particular works is framed in the music-geographical context of the Musikstadt and literary underpinnings of this topos, ranging from Ingeborg Bachmann to Hans Mayer and, once again, Thomas Mann. In analysing these texts, the methodological approach devised by Strohm, who identifies the blending of a range of urban sounds as a definition of urban space and identity, is applied. His ideas combine historical literary
analysis, musical history and urban sociology. They are rarely used in the analysis of the auditory environment.Arts and Humanities Research Council
Westfield TrustWestfield Trust Studentship
Arts and Humanities Reseach Council (AHRC
An Analysis of AI-Generated Laboratory Reports across the Chemistry Curriculum and Student Perceptions of ChatGPT
No full textAI technologies are rapidly pervading many areas of our
world.
AI-driven text generators such as ChatGPT are at the forefront of
this due to their simplicity and accessibility. Their influence on
higher education is already being observed, and perceptions among
faculty and students vary widely. We have undertaken a cross-curriculum
study of ChatGPT’s ability to generate laboratory reports.
AI-generated reports from general, organic, analytical, physical,
inorganic, and biochemistry courses were graded as if they were student
reports and analyzed for grade distributions and common strengths
and weaknesses. To further gauge ChatGPT’s current impact,
we surveyed all students in our Spring 2023 laboratory courses regarding
their awareness and use of ChatGPT. We have also laid out suggestions,
guidance, and considerations for instructors who wish to prohibit
ChatGPT use by their students as well as for those who wish to begin
incorporating this new, powerful tool into their teaching
An Analysis of AI-Generated Laboratory Reports across the Chemistry Curriculum and Student Perceptions of ChatGPT
No full textAI technologies are rapidly pervading many areas of our
world.
AI-driven text generators such as ChatGPT are at the forefront of
this due to their simplicity and accessibility. Their influence on
higher education is already being observed, and perceptions among
faculty and students vary widely. We have undertaken a cross-curriculum
study of ChatGPT’s ability to generate laboratory reports.
AI-generated reports from general, organic, analytical, physical,
inorganic, and biochemistry courses were graded as if they were student
reports and analyzed for grade distributions and common strengths
and weaknesses. To further gauge ChatGPT’s current impact,
we surveyed all students in our Spring 2023 laboratory courses regarding
their awareness and use of ChatGPT. We have also laid out suggestions,
guidance, and considerations for instructors who wish to prohibit
ChatGPT use by their students as well as for those who wish to begin
incorporating this new, powerful tool into their teaching
