Molecular and Cellular Approaches Toward Understanding Dynein-Driven Motility

Active transport is integral to organelle localization and their distribution within the cell. Kinesins, myosins and dynein are the molecular motors that drive this long range transport on the actin and microtubule cytoskeleton. Although several families of kinesins and myosins have evolved, there is only one form of cytoplasmic dynein driving active retrograde transport in cells. While dynactin is an essential co-factor for most cellular functions of dynein, the mechanistic basis for this evolutionarily well conserved interaction remains unclear. Here, I use single molecule approaches with purified dynein to reconstitute processes in vitro, and implement an optogenetic tool in neurons to further dissect regulatory mechanisms of dynein-driven transport in cells. I demonstrate for the first time, at the single molecule level, that dynactin functions as a tether to enhance the initial recruitment of dynein onto microtubules but also acts as a brake to slow the motor. I then extend this work in neurons to understand regulation of the dynein motor at the cellular level. Neurons are particulary dependent on long-range transport as organelles and macromolecules must be efficiently moved over the extended length of the axon and further, have mechanisms in place for the compartment-specific regulation of trafficking in axons and dendrites. I use a light-inducible dimerization tool to recruit motor proteins or motor adaptors to organelles in real time to examine downstream effects of organelle motility and compartment-specific regulation of motors. I find that while dynein works efficiently in both axons and dendrites, kinesins are differentially regulated in a compartment-specific manner. I further demonstrate that dynein-driven motility in neurons is largely governed by microtubule orientation and requires microtubule dynamics for efficient navigation in axons and dendrites. Together, this work sheds light on the molecular and cellular mechanisms of dynein function both in vitro and in vivo using a combination of approaches. My findings converge to a model wherein dynactin enhances the recruitment of dynein onto microtubule plus ends, leading to efficient minus-end directed motility of dynein. This becomes especially critical in neuronal growth cones and dendrites owing to the large number of highly dynamic microtubules in these compartments

Codon adaptation-based control of protein expression in C. elegans.

We present a method to control protein levels under native genetic regulation in Caenorhabditis elegans by using synthetic genes with adapted codons. We found that the force acting on the spindle in C. elegans embryos was related to the amount of the G-protein regulator GPR-1/2. Codon-adapted versions of any C. elegans gene can be designed using our web tool, C. elegans codon adapter

Optogenetic control of organelle transport using a photocaged chemical inducer of dimerization

SummaryCell polarity, growth and signaling require organelle transport by cytoskeletal motor proteins that are precisely regulated in time and space. Probing these complex, dynamic processes requires experimental techniques with comparable temporal and spatial precision. Inducible dimerization offers the ability to recruit motor proteins to organelles in living cells. Approaches include rapamycin-induced dimerization of motors and cargo-bound binding partners [1] or the recent application of the TULIP light-inducible dimerization system [2,3]. In the latter system, motor recruitment is activated by blue light, and relaxes to an OFF state in the dark within seconds. While rapid relaxation is desirable for some applications, many experiments require sustained motor recruitment. Here, we use a photocaged chemical dimerizer to achieve sustained, spatially-defined motor recruitment to individual organelles with a single pulse of light. We demonstrate the general applicability of the system by recruiting microtubule plus end-directed kinesin-1 and minus end-directed dynein motors to peroxisomes and mitochondria in HeLa cells and primary neurons, leading to alterations in organelle transport on timescales from <10 seconds to >10 minutes after photoactivation

Staged Laser Interstitial Thermal Therapy for the Surgical Treatment of Insular Glioma: a Case Series

Staged Laser Interstitial Thermal Therapy for the Surgical Treatment of Insular Glioma: a Case Series Introduction Insular gliomas pose one of the most significant challenges in neurosurgical oncology due to the complexity of the surrounding functional and microvascular anatomy. Despite current surgical and technological advancements, resection of insular gliomas can lead to potentially severe neurological morbidities. Laser Interstitial Thermal Therapy (LITT), either alone or combined with surgery, presents a less invasive cytoreductive approach for treating insular gliomas. Objective In this work we describe the first-ever reported series of patients with insular gliomas treated with staged LITT operations with or without subsequent craniotomy. Methods We reviewed a retrospective institutional database to identify patients with insular glioma who underwent staged LITT operations with or without subsequent craniotomy. From the compiled list of 11 patients, we obtained clinical, histopathological and volumetric lesion characteristics for each patient. Procedural characteristics, morbidity, overall survival (OS) and progression-free survival (PFS) were further assessed. Results From the 11 insular glioma patients included in this study the average age was 42 (SD = 12.5 years) with 8 (73%) patients that had left sided tumors and 6 (55%) patients had high grade insular gliomas. A total of 26 surgical procedures were performed with 21 ablations and 5 ablations that were followed by subsequent craniotomy. With regards to operative outcomes, the median tumor volume (cc) of our patient group was 31.5 (9.58-97) and the extent of tumor resection with laser ablation was on average 96.2% (SD = 8.5). Assessing post-operative morbidities, our group found OS to be 15.7 months (SD=10.3) and PFS to be 11.7 months (SD=7.5). Of the 21 ablation-only procedures performed, in the peri-operative period (3-5 days) neurological deficits were found in 9 (43%) post-ablation cases however there were 0 post-ablation cases with neurological deficits in the long-term (\u3e3months). Of the 5 procedures where laser ablation was followed with subsequent craniotomy, neurological deficits were found in the peri-operative period in all 5 post-resection cases (100%) and there were neurological deficits in the long-term period found in 2 of them (40%). Conclusions We present the first ever reported series of insular gliomas treated with staged LITT operations. Through conducting this study our group found that staged treatment of insular gliomas with LITT is safe, effective and a minimally invasive option that avoids the potentially severe neurological compromises associated with conventional surgical resection approaches. Usage of stages of LITT to directly target high-risk insular glioma regions presents a novel treatment approach that may facilitate the maximal safe treatment of these otherwise significantly surgically challenging lesions

Pancreatic manifestations in von Hippel–Lindau disease: A case report

Introduction: Pancreatic manifestations in Von Hippel–Lindau (VHL) disease can present as a multitude of forms, and their management can be challenging. Presentation of the case: A 66-year-old woman presented with increasing abdominal girth without other associated symptoms of nausea, vomiting, abdominal pain, weight-loss, and jaundice. Her medical and surgical histories were significant for type II diabetes, cerebral tumor resection, bilateral nephrectomies, and laser photocoagulation of retinal hemangiomas. Computed tomography (CT) of the abdomen showed a massive multi-cystic lesion in the pancreas and the patient was referred to our hepatopancreatic biliary center. Discussion: The findings on the subsequent cross-sectional MRI imaging signified pancreatic manifestations in VHL disease. Conclusion: The management of VHL disease-associated benign pancreatic cystic lesions involves interval monitoring with cross-sectional imaging for malignant changes/development