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Papers by Lani Keller

To increase students' interest in their own genomes, this computer-based laboratory lesson is... more To increase students' interest in their own genomes, this computer-based laboratory lesson is designed to be coupled with the opportunity for the students to be genotyped by the consumer sequencing company, 23andMe. Published in CourseSource

Double Helix: A Journal of Critical Thinking and Writing, 2015

Journal of Visualized Experiments, 2017

Journal of undergraduate neuroscience education : JUNE : a publication of FUN, Faculty for Undergraduate Neuroscience, 2015

In this completely digital teaching module, students interpret the results of two separate proced... more In this completely digital teaching module, students interpret the results of two separate procedures: a restriction endonuclease digestion, and a polymerase chain reaction (PCR). The first consists of matching restriction endonuclease digest protocols with images obtained from stained agarose gels. Students are given the sequence of six plasmid cDNAs, characteristics of the plasmid vector, and the endonuclease digest protocols, which specify the enzyme(s) used. Students calculate the expected lengths of digestion products using this information and free tools available on the web. Students learn how to read gels and then match their predicted fragment lengths to the digital images obtained from the gel electrophoresis of the cDNA digest. In the PCR experiment, students are given six cDNA sequences and six sets of primers. By querying NCBI BLAST, students can match the PCR fragments to the lengths of the predicted in silico PCR products. The ruse posed to students is that the gels w...

Journal of Experimental Neuroscience, 2015

Neurodegenerative diseases affect millions of people worldwide, and as the global population ages... more Neurodegenerative diseases affect millions of people worldwide, and as the global population ages, there is a critical need to improve our understanding of the molecular and cellular mechanisms that drive neurodegeneration. At the molecular level, neurodegeneration involves the activation of complex signaling pathways that drive the active destruction of neurons and their intracellular components. Here, we use an in vivo motor neuron injury assay to acutely induce neurodegeneration in order to follow the temporal order of events that occur following injury in Drosophila melanogaster. We find that sites of injury can be rapidly identified based on structural defects to the neuronal cytoskeleton that result in disrupted axonal transport. Additionally, the neuromuscular junction accumulates ubiquitinated proteins prior to the neurodegenerative events, occurring at 24 hours post injury. Our data provide insights into the early molecular events that occur during axonal and neuromuscular ...

Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology

This adaptable graduate laboratory course on protein purification offers students the opportunity... more This adaptable graduate laboratory course on protein purification offers students the opportunity to explore a wide range of techniques while allowing the instructor the freedom to incorporate their own personal research interests. The course design involves two sequential purification schemes performed in a single semester. The first part comprises the expression and purification of a recombinant GFP-binding protein from E. coli. The student-purified GFP-binding protein is then used in the second part of the course to immunoprecipitate GFP-tagged proteins, and their potential interacting partners, from cell or tissue extracts. As an example, we describe the immunoprecipitation of GFP-tagged proteins from Drosophila melanogaster larval extracts that are homologous to proteins implicated in human diseases, followed by western blotting to examine student experimental outcomes. However, the widespread availability of GFP-fusion proteins in diverse organisms enables researchers to tailo...

Physical Biology, 2011

The degree to which diffusion contributes to positioning cellular structures is an open question.... more The degree to which diffusion contributes to positioning cellular structures is an open question. Here we investigate the question of whether diffusive motion of centrin granules would allow them to interact with the mother centriole. The role of centrin granules in centriole duplication remains unclear, but some proposed functions of these granules, for example in providing preassembled centriole subunits, or by acting as unstable "pre-centrioles" that need to be captured by the mother centriole (La Terra et al., 2005), require the centrin foci to reach the mother. To test whether diffusive motion could permit such interactions in the necessary time scale, we measured the motion of centrin-containing foci in living human U2OS cells. We found that these centrin foci display apparently diffusive undirected motion. Using the apparent diffusion constant obtained from these measurements, we calculated the time scale required for diffusion to capture by the mother centrioles and found that it would greatly exceed the time available in the cell cycle. We conclude that mechanisms invoking centrin foci capture by the mother, whether as a pre-centriole or as a source of components to support later assembly, would require a form of directed motility of centrin foci that has not yet been observed.

Cytoskeleton, 2010

The functional role of centrioles or basal bodies in mitotic spindle assembly and function is cur... more The functional role of centrioles or basal bodies in mitotic spindle assembly and function is currently unclear. Although supernumerary centrioles have been associated with multipolar spindles in cancer cells, suggesting centriole number might dictate spindle polarity, bipolar spindles are able to assembly in the complete absence of centrioles, suggesting a level of centrioleindependence in the spindle assembly pathway. In this report we perturb centriole number using mutations in Chlamydomonas reinhardtii, and measure the response of the mitotic spindle to these perturbations in centriole number. Although altered centriole number increased the frequency of monopolar and multipolar spindles, the majority of spindles remained bipolar regardless of the centriole number. But even when spindles were bipolar, abnormal centriole numbers led to asymmetries in tubulin distribution, half-spindle length and spindle pole focus. Half spindle length correlated directly with number of centrioles at a pole, such that an imbalance in centriole number between the two poles of a bipolar spindle correlated with increased asymmetry between half spindle lengths. These results are consistent with centrioles playing an active role in regulating mitotic spindle length. Mutants with centriole number alteration also show increased cytokinesis defects, but these do not correlate with centriole number in the dividing cell and may therefore reflect downstream consequences of defects in preceding cell divisions.

Current Biology, 2005

During interphase, centrioles act as basal bodies by templating the formation of cilia and flagel... more During interphase, centrioles act as basal bodies by templating the formation of cilia and flagella. The im-John R. Yates III, 2 and Wallace F. Marshall 1, * portance of cilia for many developmental and physio-1 Department of Biochemistry and Biophysics logical processes [2, 3] has generated great recent in-University of California, San Francisco terest in the function of ciliary basal bodies. Several San Francisco, California 94143 human diseases that appear to involve ciliary defects-2 Department of Cell Biology for example, Bardet-Biedl syndrome-are now known The Scripps Research Institute to result from defects in basal-body-localized proteins La Jolla, California 92037 [4-6]. Given that multiple ciliary-disease genes encode basal-body-associated proteins, one potentially powerful approach for identifying additional basal-body-Summary localized ciliary-disease genes is to determine the protein composition of centrioles that have matured into Background: The centriole is one of the most enigbasal bodies. matic organelles in the cell. Centrioles are cylindrical, Direct proteomic analysis of centrioles has not premicrotubule-based barrels found in the core of the cenviously been reported. In a clever and innovative strategy trosome. Centrioles also act as basal bodies during into define cilia and centriole-related genes, comparativeterphase to nucleate the assembly of cilia and flagella. genomic analyses were previously used to determine There are currently only a handful of known centriole genes conserved in species that have cilia and flagella proteins. [4, 7]. Several of these conserved genes are known to Results: We used mass-spectrometry-based MudPIT localize to centrioles or basal bodies, consistent with (multidimensional protein identification technology) to the fact that any organism that forms cilia must also identify the protein composition of basal bodies (centricontain centrioles. However, despite the fact that one oles) isolated from the green alga Chlamydomonas reinof these datasets was named the flagellar and basalhardtii. This analysis detected the majority of known body proteome (FABP), in fact these analyses were encentriole proteins, including centrin, epsilon tubulin, tirely genomics based and did not directly address the and the cartwheel protein BLD10p. By combining proprotein composition of centrioles per se. Comparativeteomic data with information about gene expression genomics analyses are unable to detect centriolar proand comparative genomics, we identified 45 cross-valiteins, such as centrin or tubulin, which are conserved dated centriole candidate proteins in two classes. in species lacking cilia. A direct proteomic analysis of Members of the first class of proteins (BUG1-BUG27) centrioles would therefore be expected to provide comare encoded by genes whose expression correlates plementary information about centriole composition. with flagellar assembly and which therefore may play a A proteomic analysis of whole centrosomes isolated role in ciliogenesis-related functions of basal bodies. from vertebrate cells has been published [8]. However, Members of the second class (POC1-POC18) are impliit is critical to distinguish between centrioles and cencated by comparative-genomics and-proteomics trosomes, which are much larger structures composed studies to be conserved components of the centriole. of centrioles embedded within a large quantity of amor-We confirmed centriolar localization for the human hophous pericentriolar material (PCM) that is responsible mologs of four candidate proteins. Three of the crossfor nucleation of microtubules. In metazoans, centrioles validated centriole candidate proteins are encoded by are small in comparison to the large size of the entire orthologs of genes (OFD1, NPHP-4, and PACRG) implicentrosome, and one would expect that the proteins cated in mammalian ciliary function and disease, sugmaking up the centriole would be swamped out by the gesting that oral-facial-digital syndrome and nephronlarge quantity of PCM proteins if entire centrosomes ophthisis may involve a dysfunction of centrioles and/ were analyzed by proteomics. In fact, although the pubor basal bodies. lished centrosome proteome identified many proteins Conclusions: By analyzing isolated Chlamydomonas of the pericentriolar matrix, only a few known centriole basal bodies, we have been able to obtain the first reproteins were identified in that study; for example, epsiported proteomic analysis of the centriole. lon tubulin was not found. In contrast, Chlamydomonas basal bodies represent essentially naked centrioles, Introduction without any appreciable PCM. Therefore, a proteomic analysis of Chlamydomonas basal bodies should allow Centrioles are composed of nine triplet microtubules identification of bona fide centriole proteins to a higher arranged into a cylinder with a 250 nm diameter [1]. degree of sensitivity than previous studies. Moreover, The function of centrioles, and the mechanism of their because eukaryotic basal bodies template flagellar asduplication, remains unclear. Much of the aura of myssembly during interphase and also act as centrioles in tery that surrounds centrioles stems from the fact that mitosis (as first recognized in the 19th century by Henthe protein composition of centrioles is largely unneguy and von Lenhossék), analysis of isolated Chlaknown. mydomonas basal bodies should reveal both proteins found within the mitotic centriole as well as basalbody-specific proteins involved in ciliary assembly.

Biochemistry and Molecular Biology Education, 2017

Methods in molecular biology (Clifton, N.J.), 2008

Centrioles are barrel-shaped cytoskeletal organelles composed of nine triplet microtubules blades... more Centrioles are barrel-shaped cytoskeletal organelles composed of nine triplet microtubules blades arranged in a pinwheel-shaped array. Centrioles are required for recruitment of pericentriolar material (PCM) during centrosome formation, and they act as basal bodies, which are necessary for the outgrowth of cilia and flagella. Despite being described over a hundred years ago, centrioles are still among the most enigmatic organelles in all of cell biology. To gain molecular insights into the function and assembly of centrioles, we sought to determine the composition of the centriole proteome. Here, we describe a method that allows for the isolation of virtually "naked" centrioles, with little to no obscuring PCM, from the green alga, Chlamydomonas. Proteomic analysis of this material provided evidence that multiple human disease gene products encode protein components of the centriole, including genes involved in Meckel syndrome and Oral-Facial-Digital syndrome. Isolated cen...

Molecular Biology of the Cell, 2009

Centrioles are intriguing cylindrical organelles composed of triplet microtubules. Proteomic data... more Centrioles are intriguing cylindrical organelles composed of triplet microtubules. Proteomic data suggest that a large number of proteins besides tubulin are necessary for the formation and maintenance of a centriole's complex structure. Expansion of the preexisting centriole proteome from the green alga Chlamydomonas reinhardtii revealed additional human disease genes, emphasizing the significance of centrioles in normal human tissue homeostasis. We found that two classes of ciliary disease genes were highly represented among the basal body proteome: cystic kidney disease (especially nephronophthisis) syndromes, including Meckel/Joubert-like and oral-facial-digital syndrome, caused by mutations in CEP290, MKS1, OFD1, and AHI1/Jouberin proteins and cone-rod dystrophy syndrome genes, including UNC-119/HRG4, NPHP4, and RPGR1. We further characterized proteome of the centriole (POC) 1, a highly abundant WD40 domain-containing centriole protein. We found that POC1 is recruited to na...

Neural Regeneration Research, 2016

Neuron, 2011

We provide evidence for a prodegenerative, glialderived signaling framework in the Drosophila neu... more We provide evidence for a prodegenerative, glialderived signaling framework in the Drosophila neuromuscular system that includes caspase and mitochondria-dependent signaling. We demonstrate that Drosophila TNF-a (eiger) is expressed in a subset of peripheral glia, and the TNF-a receptor (TNFR), Wengen, is expressed in motoneurons. NMJ degeneration caused by disruption of the spectrin/ankyrin skeleton is suppressed by an eiger mutation or by eiger knockdown within a subset of peripheral glia. Loss of wengen in motoneurons causes a similar suppression providing evidence for glial-derived prodegenerative TNF-a signaling. Neither JNK nor NFkb is required for prodegenerative signaling. However, we provide evidence for the involvement of both an initiator and effector caspase, Dronc and Dcp-1, and mitochondrial-dependent signaling. Mutations that deplete the axon and nerve terminal of mitochondria suppress degeneration as do mutations in Drosophila Bcl-2 (debcl), a mitochondria-associated protein, and Apaf-1 (dark), which links mitochondrial signaling with caspase activity in other systems. Neuron Glial-Derived Prodegenerative Signaling