Minisymposium: Molecular Motors: Stopped or Slowed by Their Tracks (2012)

Department of Biochemistryand Molecular Pharmacology, UMass Medical School/HHMI, Worcester,MA, USA.Excision of introns from pre-mRNAs is mediated by the spliceosome, a large,dynamiccomplexconsistingofﬁvesmallribonucleoproteinparticles(snRNPs)andscoresofassociatedproteins.Currentunderstandingofspliceosomeassem-bly is based largely on the procession of stable complexes that can be resolvedfrom in vitro splicing reactions. Such ensemble experiments have suggesteda highly ordered, linear assembly pathway in which initial binding of U1snRNPto the5’ splicesite is followedbystable U2 associationwith thebranchsite and subsequent U4/U5/U6 tri-snRNP and Nineteen Complex (NTC) addi-tion to form the fully assembled spliceosome. Previously unknown, however,werethedetailedforwardandreversekineticsofeachassemblystep,theextentto which branched and/or dead-end assembly pathways exist, and whether ornot different introns utilize the same or alternate assembly pathway(s). Weare now addressing these questions by combining yeast genetic engineering,chemical biology, and multi-wavelengthﬂuoresence microscopy to follow as-sembly of single spliceosomes in real time. Because no protein puriﬁcationor reconstitution is required for such Colocalization Single Molecule Spectros-copy (CoSMoS), this experimental strategy should prove widely useful formechanistic analysisof many other macromolecular machines in environmentsapproaching the complexity of living cells.3100-SympChromatin Dynamics: At the Source of RNA ProductionChristophe Lavelle.National Museum of Natural History, Paris, France.Through its local heterogeneities and transient structural changes resultingfrom chemical modiﬁcations and physical constraints imposed by numerousactors in vivo, chromatin dynamics inﬂuences (and is inﬂuenced by) DNAtranscription, both at the initiation and elongation stages.While transcription events often correlate with large chromatin movements,RNApolymeraseaccesstoitstargetsequenceimpliessomenucleosomedynam-ics (potentially mediated bychromatin remodeling factors)and its subsequent trackingalong the DNA template im-poses some drastic topologicalandstructuralchangesthatprop-agateinthetranscribedchroma-tin domain. Experiments andmodels aimed at decipheringthe key features of chromatindynamics and topology upontranscription will be presented.In a second part of the talk, we will shift from eukaryotes to prokaryotes andshow some recent data on ncRNA self-assembly, which role (and potential oc-currence in eukaryotes) will be questioned.3101-SympBiophysics of the Interaction of HIV with Mucus Barriers during SexualTransmissionThomas Hope.Department of Cellular & Molecular Biology, Northwestern University,Chicago, IL, USA.During sexual transmission, HIV must overcome mucosal barriers to reach un-derlying target cells. The epithelial barrier function of the female reproductivetract is further enhanced by a protective layer of cervical mucus (CM). It is be-lieved that antibodies associated with mucus barriers of the gut can have anti-microbial activity, and so we explored the possible role that antibodies whichcan bind to virions might alter particle transport through mucus. To character-ize the effects of HIV-speciﬁc antibodies on viral diffusion in CM, we utilizedtwo red and green ﬂuorescently tagged HIV types: wildtype enveloped virus(WT-Gag-mCherry) and HIV devoid of envelope proteins, (DEnv-Gag-GFP)which allows for simultaneous visualization of both virus types in CM. Forourassay,bothvirustypesweremixedatequalconcentrationsandeitheraddeddirectly to CM or incubated with HIV-speciﬁc antibodies prior to being addedto CM. Particle tracking software was used to determine particle position andmeasure the mean squared displacement (MSD), a standard measure of micro-scopic motion. We found that using anti-MHC class I antibodies, which bindepitopes found on WT and DEnv virus, decrease the movement of both virustypes. In addition, neutralizing and non-neutralizing anti-gp41 and anti-gp120 antibodies speciﬁcally impaired the mobility of WT virus when com-pared to DEnv virus or when compared to virions that received no antibodytreatment. In addition, we observed greater inhibition of HIV transport inCM with multimeric anti-Env antibodies when compared to monomeric anti-bodies. Our studies reveal that virus binding antibodies can slow the transportofHIVwithinthemucuscoatingofthefemalereproductivetract.Thissuggeststhat a vaccine that generates broadly binding antibodies could potentially pre-vent viral interactions with target cells thus offering protection against produc-tive infection.