Self-crowding of AMPA receptors in the excitatory postsynaptic density can effectuate anomalous receptor sub-diffusion.

AMPA receptors (AMPARs) and their associations with auxiliary transmembrane proteins are bulky structures with large steric-exclusion volumes. Hence, self-crowding of AMPARs, depending on the local density, may affect their lateral diffusion in the postsynaptic membrane as well as in the highly crowded postsynaptic density (PSD) at excitatory synapses. Earlier theoretical studies considered only the roles of transmembrane obstacles and the AMPAR-binding submembranous scaffold proteins in shaping receptor diffusion within PSD. Using lattice model of diffusion, the present study investigates the additional impacts of self-crowding on the anomalousity and effective diffusion coefficient (Deff) of AMPAR diffusion. A recursive algorithm for avoiding false self-blocking during diffusion simulation is also proposed. The findings suggest that high density of AMPARs in the obstacle-free membrane itself engenders strongly anomalous diffusion and severe decline in Deff. Adding transmembrane obstacles to the membrane accentuates the anomalousity arising from self-crowding due to the reduced free diffusion space. Contrarily, enhanced AMPAR-scaffold binding, either through increase in binding strength or scaffold density or both, ameliorates the anomalousity resulting from self-crowding. However, binding has differential impacts on Deff depending on the receptor density. Increase in binding causes consistent decrease in Deff for low and moderate receptor density. For high density, binding increases Deff as long as it reduces anomalousity associated with intense self-crowding. Given a sufficiently strong binding condition when diffusion acquires normal behavior, further increase in binding causes decrease in Deff. Supporting earlier experimental observations are mentioned and implications of present findings to the experimental observations on AMPAR diffusion are also drawn

Glucose-sensing in the hypothalamic arcuate nucleus : electrophysiological and mathematical studies

1. Energy homeostasis requires the co-ordination of several metabolic fluxes at the systemic level among various peripheral organs and the central nervous system. A breakdown of these metabolic fluxes can lead to hyperglycaemia. An explicit representation of this complex dynamical system can aid the interpretation of diagnostic tests and help formulate therapeutic interventions. Hence, mathematical models could constitute a valuable clinical tool in the management of hyperglycaemia associated with diabetes. However, a very large range of such models is available, making a judicious choice difficult. To better inform this choice, the most important models published to date are presented in a uniform format, discussing similarities and differences in terms of the decisions faced by modellers. We review models for glucostasis, based on the glucose-insulin feedback control loop, and consider extensions to long-term energy balance, dislipidæmia and obesity. 2. Whole-cell patch-clamp recording techniques were used in isolated hypothalamic brain slice preparations to investigate and compare the electrophysiological properties of arcuate nucleus (ARC) neurones from fed and fasted rats, including a fed control group housed as fasted animals. Subthreshold active conductances were differentially expressed in ARC neurones including: anomalous inward rectification (Ian), A-like transient outward rectification (IA), time and voltage-dependent inward rectification (Ih) and T-type calcium-like conductance. Significant differences in active and passive subthreshold membrane properties of ARC neurones were observed between the three groups, including: changes in magnitude of IA and Ih, action potential duration, membrane time-constant (tau), neuronal input resistance and spontaneous activity. Furthermore, both housing and fasting conditions affected electrophysiological properties of rat ARC neurones, suggesting both stress and fasting can modify electrophysiological properties of ARC neurones. 3. The effects of intracellular adenosine triphosphate (ATP) on neuronal excitability of ARC neurones were investigated and compared between fed and fasted rats. This was performed by manipulating extracellular glucose levels from 2.0 to 0.0 mM whilst intracellular ATP was manipulated by changing the levels in the patch pipette solution (0.0, 1.0, 2.0, 5.0 and 10.0 mM). The level of ATP required to maintain resting membrane potential and glucose-sensing capability of ARC neurones was determined. Data from this study suggests 1.0 mM and 5.0 mM ATP for fasted and fed rats, respectively, were appropriate levels for maintaining electrophysiological and glucose-sensing integrity of these neurones. Hence levels of or sensitivity to ATP appears subject to modulation depending on the energy status of organism. 4. Glucose-sensing neurones and associated underlying mechanisms in ARC neurones in both fed and fasted states were studied. Extracellular glucose levels (2.0 – 0.2 – 0.5 – 1.0 – 2.0 – 5.0 mM) were manipulated with appropriate intracellular ATP levels determined as outlined above. Three types of glucose-sensing neurone were identified: glucose-excited (GE) neurones, glucose-inhibited (GI) neurones and glucose-rapidly adapting (GRP) neurones. The proportions of these three groups of neurones varied between fed and 24-hour fasted rats. Changing energy status, fasting, also appeared to affect the sensitivity of glucose-sensing neurones, i.e. the threshold levels of glucose they detect. GE neurones operated through ATP-sensitive potassium (KATP) channel-dependent mechanisms in fed and fasted rats and through a chloride-dependent mechanism in fed rats. GI neurones detect changes in glucose levels through chloride and/or non-selective cation conductance-dependent mechanisms. Finally a potential mechanism of GRA neurones may be through transient opening of chloride conductances. Further work is required to confirm the ionic mechanisms of these glucose-sensing neurones. 5. Analysis of responses observed in ARC glucose-sensing neurones was also examined using a mathematical approach. An empirical sigmoid response function was assumed to describe the time course of the transition in neuronal activity and the integral of this function was fitted to cumulative action potential numbers to characterise the response dynamics. Four parameters were estimated for each transition, employing the least-squares criterion; the activity prior to and at the end of each change in extracellular glucose concentration, the half sigmoid time before the neuronal activity changes following a step change in extracellular glucose and the duration of the transition. Statistical tests revealed a statistically detectable difference in response delay of GE neurones following step changes in glucose levels between fed and 24-hour fasted rats. In addition, in fed rats, statistical test revealed that GE neurones required a significantly shorter delay time in changing their activity but longer change-over times than GI neurones. In 24-hour fasted rats, only the difference in activity of neurones at the start and at the end of glucose application was found to be statistically significant difference between GE and GI neurones. Further work is required to confirm these data.EThOS - Electronic Theses Online ServiceThailand. Ratthasaphā [Thailand. Parliament]GBUnited Kingdo

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin