Cytoplasmic dynein binding, run length, and velocity are guided by long-range electrostatic interactions

Dyneins are important molecular motors involved in many essential biological processes, including cargo transport along microtubules, mitosis, and in cilia. Dynein motility involves the coupling of microtubule binding and unbinding to a change in the configuration of the linker domain induced by ATP hydrolysis, which occur some 25 nm apart. This leaves the accuracy of dynein stepping relatively inaccurate and susceptible to thermal noise. Using multi-scale modeling with a computational focusing technique, we demonstrate that the microtubule forms an electrostatic funnel that guides the dynein’s microtubule binding domain (MTBD) as it finally docks to the precise, keyed binding location on the microtubule. Furthermore, we demonstrate that electrostatic component of the MTBD’s binding free energy is linearly correlated with the velocity and run length of dynein, and we use this linearity to predict the effect of mutating each glutamic and aspartic acid located in MTBD domain to alanine. Lastly, we show that the binding of dynein to the microtubule is associated with conformational changes involving several helices, and we localize flexible hinge points within the stalk helices. Taken all together, we demonstrate that long range electrostatic interactions bring a level of precision to an otherwise noisy dynein stepping process

Release mechanism of octadecyl rhodamine B chloride from Au nanorods by ultrafast laser pulses

We investigated the release of octadecyl rhodamine B chloride (R[subscript 18]) loaded onto cetyltrimethylammonium bromide (CTAB) coated gold nanorods (NR) by pulsed ultrafast laser excitation. R[subscript 18] intercalates into the hydrophobic CTAB bilayer on the NR surface and can exchange on and off the NR with free CTAB micelles in solution. We find that laser excitation accelerates the rate of both R[subscript 18] release from the NR and R[subscript 18] binding to the NR with increasing fluence. However, at laser fluences >220 μJ/cm[superscript 2] thermal degradation of the R[subscript 18] dominates. We also find that the concentration of CTAB, particularly around the critical micelle concentration, strongly influences the release and binding rates

Physical and practical limits of a biomolecular control system using nanoparticles and electromagnetic field irradiation

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2010.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references (p. 195-210).Many nanometer length scale engineering applications of mechanics and biology including computation, sensing, self-assembly, transport, and molecular machine design take advantage of natural biomolecular machinery. Further development of these technologies requires direct, external biomolecular control. This thesis proposes a simple control technique: a biomolecular \on/o" activity switch in which metallic nanoparticles (NPs) are conjugated to target biomolecules and irradiated with an electromagnetic field. Due to their unique physical properties, the NPs specifically absorb the field's energy. They convert the energy to heat, and then they transport it to the conjugated target biomolecules. The heat affects a change in the targeted biomolecules, selectively actuating their activity. This thesis is on the mechanisms by which both ultrafast pulsed laser irradiation and radio frequency alternating magnetic fields (RFMFs) can be used as energy sources for the proposed biomolecular activity switch. The thesis reports on the quantification of a fs-pulsed laser triggered release mechanism that actuates activity of the molecules released from NPs. The release mechanism is governed by NP surface chemistry. The operating window for the critical parameters governing release including NP properties and laser fluence is defined. The thesis also reports on transmission pump-probe experiments that show the thermal interface conductance (G) of NPs is critical to nanoscale thermal transport, and that G is a strong function of the NP's surface chemistry. The thesis concludes that an ultrafast pulsed laser actuated biomolecular activity switch is feasible if the critical parameters are carefully controlled. However, experimental studies revealed that using RFMFs in this biomolecular activity switching technique is not feasible. These results are validated by theoretical and analytical studies of nanoscale heat generation and transport in the system. The results presented in this thesis have implications on the design of the biomolecular activity switch, and many of the results are also applicable to other nanoscale thermal applications including hyperthermia cancer treatments and triggered drug delivery techniques.by Joshua Daniel Alper.Ph.D

E-hooks provide guidance and a soft landing for the microtubule binding domain of dynein

Macromolecular binding is a complex process that involves sensing and approaching the binding partner, adopting the proper orientation, and performing the physical binding. We computationally investigated the role of E-hooks, which are intrinsically disordered regions (IDRs) at the C-terminus of tubulin, on dynein microtubule binding domain (MTBD) binding to the microtubule as a function of the distance between the MTBD and its binding site on the microtubule. Our results demonstrated that the contacts between E-hooks and the MTBD are dynamical; multiple negatively charted patches of amino acids on the E-hooks grab and release the same positively charged patches on the MTBD as it approaches the microtubule. Even when the distance between the MTBD and the microtubule was greater than the E-hook length, the E-hooks sensed and guided MTBD via long-range electrostatic interactions in our simulations. Moreover, we found that E-hooks exerted electrostatic forces on the MTBD that were distance dependent; the force pulls the MTBD toward the microtubule at long distances but opposes binding at short distances. This mechanism provides a “soft-landing” for the MTBD as it binds to the microtubule. Finally, our analysis of the conformational states of E-hooks in presence and absence of the MTBD indicates that the binding process is a mixture of the induced-fit and lock-and-key macromolecular binding hypotheses. Overall, this novel binding mechanism is termed “guided-soft-binding” and could have broad-reaching impacts on the understanding of how IDRs dock to structured proteins

Forces and Disease: Electrostatic force differences caused by mutations in kinesin motor domains can distinguish between disease-causing and non-disease-causing mutations

The ability to predict if a given mutation is disease-causing or not has enormous potential to impact human health. Typically, these predictions are made by assessing the effects of mutation on macromolecular stability and amino acid conservation. Here we report a novel feature: the electrostatic component of the force acting between a kinesin motor domain and tubulin. We demonstrate that changes in the electrostatic component of the binding force are able to discriminate between disease-causing and non-disease-causing mutations found in human kinesin motor domains using the receiver operating characteristic (ROC). Because diseases may originate from multiple effects not related to kinesin-microtubule binding, the prediction rate of 0.843 area under the ROC plot due to the change in magnitude of the electrostatic force alone is remarkable. These results reflect the dependence of kinesin’s function on motility along the microtubule, which suggests a precise balance of microtubule binding forces is required

Light chain 2 is a Tctex-type related axonemal dynein light chain that regulates directional ciliary motility in \u3ci\u3eTrypanosoma brucei\u3c/i\u3e

Flagellar motility is essential for the cell morphology, viability, and virulence of pathogenic kinetoplastids. Trypanosoma brucei flagella beat with a bending wave that propagates from the flagellum’s tip to its base, rather than base-to-tip as in other eukaryotes. Thousands of dynein motor proteins coordinate their activity to drive ciliary bending wave propagation. Dynein-associated light and intermediate chains regulate the biophysical mechanisms of axonemal dynein. Tctex-type outer arm dynein light chain 2 (LC2) regulates flagellar bending wave propagation direction, amplitude, and frequency in Chlamydomonas reinhardtii. However, the role of Tctex-type light chains in regulating T. brucei motility is unknown. Here, we used a combination of bioinformatics, in-situ molecular tagging, and immunofluorescence microscopy to identify a Tctex-type light chain in the procyclic form of T. brucei (TbLC2). We knocked down TbLC2 expression using RNAi in both wild-type and FLAM3, a flagellar attachment zone protein, knockdown cells and quantified TbLC2’s effects on trypanosome cell biology and biophysics. We found that TbLC2 knockdown reduced the directional persistence of trypanosome cell swimming, induced an asymmetric ciliary bending waveform, modulated the bias between the base-to-tip and tip-to-base beating modes, and increased the beating frequency. Together, our findings are consistent with a model of TbLC2 as a down-regulator of axonemal dynein activity that stabilizes the forward tip-to-base beating ciliary waveform characteristic of trypanosome cells. Our work sheds light on axonemal dynein regulation mechanisms that contribute to pathogenic kinetoplastids’ unique tip-to-base ciliary beating nature and how those mechanisms underlie dynein-driven ciliary motility more generally

The word landscape of the non-coding segments of the Arabidopsis thaliana genome

<p>Abstract</p> <p>Background</p> <p>Genome sequences can be conceptualized as arrangements of motifs or words. The frequencies and positional distributions of these words within particular non-coding genomic segments provide important insights into how the words function in processes such as mRNA stability and regulation of gene expression.</p> <p>Results</p> <p>Using an enumerative word discovery approach, we investigated the frequencies and positional distributions of all 65,536 different 8-letter words in the genome of <it>Arabidopsis thaliana</it>. Focusing on promoter regions, introns, and 3' and 5' untranslated regions (3'UTRs and 5'UTRs), we compared word frequencies in these segments to genome-wide frequencies. The statistically interesting words in each segment were clustered with similar words to generate motif logos. We investigated whether words were clustered at particular locations or were distributed randomly within each genomic segment, and we classified the words using gene expression information from public repositories. Finally, we investigated whether particular sets of words appeared together more frequently than others.</p> <p>Conclusion</p> <p>Our studies provide a detailed view of the word composition of several segments of the non-coding portion of the <it>Arabidopsis </it>genome. Each segment contains a unique word-based signature. The respective signatures consist of the sets of enriched words, 'unwords', and word pairs within a segment, as well as the preferential locations and functional classifications for the signature words. Additionally, the positional distributions of enriched words within the segments highlight possible functional elements, and the co-associations of words in promoter regions likely represent the formation of higher order regulatory modules. This work is an important step toward fully cataloguing the functional elements of the <it>Arabidopsis </it>genome.</p

Leveraging high-resolution 7-tesla MRI to derive quantitative metrics for the trigeminal nerve and subnuclei of limbic structures in trigeminal neuralgia

Background: Trigeminal Neuralgia (TN) is a chronic neurological disease that is strongly associated with neurovascular compression (NVC) of the trigeminal nerve near its root entry zone. The trigeminal nerve at the site of NVC has been extensively studied but limbic structures that are potentially involved in TN have not been adequately characterized. Specifically, the hippocampus is a stress-sensitive region which may be structurally impacted by chronic TN pain. As the center of the emotion-related network, the amygdala is closely related to stress regulation and may be associated with TN pain as well. The thalamus, which is involved in the trigeminal sensory pathway and nociception, may play a role in pain processing of TN. The objective of this study was to assess structural alterations in the trigeminal nerve and subregions of the hippocampus, amygdala, and thalamus in TN patients using ultra-high field MRI and examine quantitative differences in these structures compared with healthy controls. Methods: Thirteen TN patients and 13 matched controls were scanned at 7-Tesla MRI with high resolution, T1- weighted imaging. Nerve cross sectional area (CSA) was measured and an automated algorithm was used to segment hippocampal, amygdaloid, and thalamic subregions. Nerve CSA and limbic structure subnuclei volumes were compared between TN patients and controls. Results: CSA of the posterior cisternal nerve on the symptomatic side was smaller in patients (3.75mm2) compared with side-matched controls (5.77mm2, p = 0.006). In TN patients, basal subnucleus amygdala volume (0.347mm3) was reduced on the symptomatic side compared with controls (0.401mm3, p = 0.025) and the paralaminar subnucleus volume (0.04mm3) was also reduced on the symptomatic side compared with controls (0.05mm3, p = 0.009). The central lateral thalamic subnucleus was larger in TN patients on both the symptomatic side (0.033mm3) and asymptomatic side (0.035mm3), compared with the corresponding sides in controls (0.025mm3 on both sides, p = 0.048 and p = 0.003 respectively). The inferior and lateral pulvinar thalamic subnuclei were both reduced in TN patients on the symptomatic side (0.2mm3 and 0.17mm3 respectively) compared to controls (0.23mm3, p = 0.04 and 0.18 mm3, p = 0.04 respectively). No significant findings were found in the hippocampal subfields analyzed. Conclusions: These findings, generated through a highly sensitive 7 T MRI protocol, provide compelling support for the theory that TN neurobiology is a complex amalgamation of local structural changes within the trigeminal nerve and structural alterations in subnuclei of limbic structures directly and indirectly involved in nociception and pain processing