From PhD to Life

By Craig W. Stropkay, (PhD ’13, Molecular and Cell Biology, Ren lab)

Reach for the stars, they said. You should definitely go get your PhD, you’d be great for it, they said. Well, I guess they did have a point. Pursuing my doctorate degree in Molecular Biology at Brandeis was definitely one of the most challenging things that I have ever had to do in my life. I could spend hours telling you about the long hours I spent trying to construct my dissertation or the countless nights that I had to wake up and drive into the lab from Medford just to “feed” my cells — but that’s not the point of this article. I want to talk about something that I wish was more openly discussed when I first started my journey towards pursuing a PhD. Something that I believe is important to anyone who is currently working their way towards earning their doctoral degree: a job.

Now I know what you may be thinking: why would I need to worry about a job when I know I will continue onto a postdoc and then a tenure-track academic post? Isn’t that what everyone does? That is precisely my point. Don’t get me wrong: there is absolutely nothing wrong with continuing a career in academia upon completion of your doctorate. It takes a lot of patience, skill, and dedication to remain in the field after you have literally spent years becoming an expert in everything dealing with Life Science. Maybe you’ve considered going that route, feeling that your choices are limited. Many people believe that apart from academia, their only “alternative” option is to go into industry and work in biotech or pharma.

Image from Naturejobs article

[Read more…]

The Benefits of Middle Age

Almost all our cells harbor a sensory organelle called the primary cilium, homologous to the better known flagella found in protists. Some of these cilia can beat and allow the cell to move (eg. in sperm), or move fluid (eg. cerebrospinal fluid) around them. However, other specialized cilia such as those found in photoreceptor cells and in our olfactory neurons function solely as sensory organelles, providing the primary site for signal reception and transduction. The vast majority of our somatic cells display a short and simple rod-like cilium that plays crucial roles during development and in adulthood. For instance, during development, they are essential for transducing critical secreted developmental signals such as Sonic hedgehog that is required for the elaboration of cell types in almost every tissue (eg. in brain, bones, muscles, skin). In adults, cilia are required for normal functioning of our kidneys, and primary cilia in hypothalamic neurons have been shown to regulate hunger and satiety.

Given their importance, it is not surprising that defects in cilia structure and function lead to a whole host of diseases ranging from severe developmental disorders and embryonic lethality to hydrocephalus (fluid accumulation in the brain), infertility, obesity, blindness, and polycystic kidney among others. Often these diseases manifest early in development resulting in prenatal death or severe disability, but milder ciliary dysfunction leads to disease phenotypes later in life.

Much is now known about how cilia are formed and how they function during development. However, surprisingly, how aging affects cilia, and possibly the severity of cilia-related diseases, is not well studied. A new study by postdocs Astrid Cornils and Ashish Maurya, and graduate student Lauren Tereshko from Piali Sengupta’s laboratory, and collaborators at University College Dublin and University of Iowa, begins to address this question using the microscopic roundworm C. elegans (pictured below). These worms display cilia on a set of sensory neurons; these cilia are built by mechanisms that are similar to those in other animals including in humans. Worms have a life span of about 2-3 weeks, thereby making the study of how aging affects cilia function quite feasible.


They find that cilia structure is somewhat altered in extreme old age in control animals. However, unexpectedly, when they looked at animals carrying mutations that lead to human ciliary diseases, the severely defective cilia seen in larvae and young adults displayed a partial but significant recovery during middle-age, a period associated with declining reproductive function. They went on to show that the heat-shock response and the ubiquitin-proteasome system, two major pathways required for alleviating protein misfolding stress in aging and neurodegenerative diseases, are essential for this age-dependent cilia recovery in mutant animals. This restoration of cilia function is transient; cilia structure becomes defective again in extreme old age. These results suggest that increased function of protein quality control mechanisms during middle age can transiently suppress the effects of some mutations in cilia genes, and raise the possibility that these findings may help guide the design of therapeutic strategies to target specific ciliary diseases. Some things can improve with aging!

Sleep suppresses brain rebalancing

Why humans and other animals sleep is one of the remaining deep mysteries of physiology. One prominent theory in neuroscience is that sleep is when the brain replays memories “offline” to better encode them (“memory consolidation”). A prominent and competing theory is that sleep is important for re-balancing activity in brain networks that have been perturbed during learning while awake. Such “rebalancing” of brain activity involves homeostatic plasticity mechanisms that were first discovered at Brandeis University, and have been thoroughly studied by a number of Brandeis labs including the Turrigiano lab. Now, a study from the Turrigiano lab just published in the journal Cell shows that these homeostatic mechanisms are indeed gated by sleep and wake, but in the opposite direction from that theorized previously: homeostatic brain rebalancing occurs exclusively when animals are awake, and is suppressed by sleep. These findings raise the intriguing possibility that different forms of brain plasticity – for example those involved in memory consolidation and those involved in homeostatic rebalancing – must be temporally segregated from each other to prevent interference.


The requirement that neurons carefully maintain an average firing rate, much like the thermostat in a house senses and maintains temperature, has long been suggested by computational work. Without homeostatic (“thermostat-like”) control of firing rates, models of neural networks cannot learn and drift into states of epilepsy-like saturation or complete quiescence. Much of the work in discovering and describing candidate mechanisms continues to be conducted at Brandeis. In 2013, the Turrigiano Lab provided the first ­in vivo evidence for firing rate homeostasis in the mammalian brain: lab members recorded the activity of individual neurons in the visual cortex of freely behaving rat pups for 8h per day across a nine-day period during which vision through one eye was occluded. The activity of neurons initially dropped, but over the next 4 days, firing rates came back to basal levels despite the visual occlusion. In essence, these experiments confirmed what had long been suspected – the activity of neurons in intact brains is indeed homeostatically governed.

Due to the unique opportunity to study a fundamental mechanism of brain plasticity in an unrestrained animal, the lab has been probing the possibility of an intersection between an animal’s behavior and homeostatic plasticity. In order to truly evaluate possible circadian and behavioral influences on neuronal homeostasis, it was necessary to capture the entire 9-day experiment, rather than evaluate snapshots of each day. For this work, the Turrigiano Lab had to find creative computational solutions to recording many terabytes of data necessary to follow the activity of single neurons without interruption for more than 200 hours. Ultimately, these data revealed that the homeostatic regulation of neuronal activity in the cortex is gated by sleep and wake states. In a surprising and unpredicted twist, the homeostatic recovery of activity occurred almost exclusively during periods of activity and was inhibited during sleep. Prior predictions either assumed no role for behavioral state, or that sleeping would account for homeostasis. Finally, the lab established evidence for a causal role for active waking by artificially enhancing natural waking periods during the homeostatic rebound. When animals were kept awake, homeostatic plasticity was further enhanced.

This finding opens doors onto a new field of understanding the behavioral, environmental, and circadian influences on homeostatic plasticity mechanisms in the brain. Some of the key questions that immediately beg to be answered include:

  • What it is about sleep that precludes the expression of homeostatic plasticity?
  • How is it possible that mechanisms requiring complex patterns of transcription, translation, trafficking, and modification can be modulated on the short timescales of behavioral state-transitions in rodents?
  • And finally, how generalizable is this finding? As homeostasis is bidirectional, does a shift in the opposite direction similarly require wake or does the change in sign allow for new rules in expression?

Authors on the paper include postdoctoral fellow Keith Hengen, Neuroscience grad student Alejandro Torrado Pachedo, and Neuroscience undergraduate James McGregor ’14 (now in grad school at Emory).

Hengen KB, Torrado Pacheco A, McGregor JN, Van Hooser SD, Turrigiano GG. Neuronal Firing Rate Homeostasis is Inhibited by Sleep and Promoted by Wake. Cell. 2016.

Introduction to Microfluidics Technology – June 13-17, 2016

2016 MRSEC Summer Course Announcement

Registration for our annual, one-week summer course, “Introduction to Microfluidics Technology” at Brandeis University, near Boston, MA, is now open. The application deadline is March 31, 2016.

Introduction to Microfluidics Technology is a hands-on laboratory course sponsored by the National Science Foundation’s Bioinspired Soft Materials Research Science and Engineering Center (MRSEC) at Brandeis. It will be offered during the week of June 13 ‐ 17, 2016. The course is intended for graduate students, post docs, faculty, and industrial scientists/engineers interested in utilizing microfluidic technology in their work, both in the physical and life sciences. The course does not assume any specific prerequisites.

“Introduction to Microfluidics Technology” (June 13 – 17, 2016)
will be taught by Dr. Nathan Tompkins.

The $750 fee covers course tuition, housing in double-occupancy rooms, and breakfast/lunch/coffee from Monday through Friday. Single rooms are not available. Local students who do not need housing will pay a non-resident fee of $500 (cash and check only please).

More information is available.

Lipids hit a “sweet spot” to direct cellular membrane remodeling.

Lipid membrane reshaping is critical to many common cellular processes, including cargo trafficking, cell motility, and organelle biogenesis. The Rodal lab studies how dynamic membrane remodeling is achieved by the active interplay between lipids and proteins. Recent results, published in Cell Reports, demonstrate that for the membrane remodeling protein Nervous Wreck (Nwk), intramolecular autoregulation and membrane charge work together in surprising ways to restrict remodeling to a limited range of lipid compositions.

F-BAR (Fes/Cip4 homology Bin/Amphiphysin/Rvs) domain family proteins are important mediators of membrane remodeling events. The F-BAR domain forms a crescent-shaped α-helical dimer that interacts with and deforms negatively charged membrane phospholipids by assembling into higher-order scaffolds. In this paper, Kelley et al. have shown that the neuronal F-BAR protein Nwk is autoregulated by its C-terminal SH3 domains, which interact directly with the F-BAR domain to inhibit membrane binding. Until now, the dogma in the field has been that increasing concentrations of negatively charged lipids would increase Nwk membrane binding, and thus would induce membrane deformation.

Surprisingly, Kelley et al. found that autoregulation does not mediate this kind of simple “on-off” switch for membrane remodeling. Instead, increasing the concentration of negatively charged lipids increases membrane binding, but inhibits F-BAR membrane deforming activities (see below). Using a combination of in vitro assays and single particle electron microscopy, they found that the Nwk F-BAR domain efficiently assembles into higher-order structures and deforms membranes only within “sweet spot” of negative membrane charge, and that autoregulation elevates this range. The implication of this work is that autoregulation could either reduce membrane binding or promote higher-order assembly, depending on local cellular membrane composition. This study suggests a significant role for the regulation of membrane composition in remodeling.

Brandeis authors on the study included Molecular and Cell Biology graduate students Charlotte Kelley and Shiyu Wang, staff member Tania Eskin, and undergraduate Emily Messelaar ’13 from the Rodal lab; postdoctoral fellow Kangkang Song, Associate Professor of Biology Daniela Nicastro (currently at UT Southwestern), and Associate Professor of Physics Michael Hagan.

Kelley CF, Messelaar EM, Eskin TL, Wang S, Song K, Vishnia K, Becalska AN, Shupliakov O, Hagan MF, Danino D, Sokolova OS, Nicastro D, Rodal AA. Membrane Charge Directs the Outcome of F-BAR Domain Lipid Binding and Autoregulation. Cell reports. 2015;13(11):2597-609.

Visualizing a protein decision complex in actin filament length control

Seen at the Gelles Lab Little Engine Shop blog this week, commentary on a new paper in Nature Communicationspublished in collaboration with the Goode Lab and researchers from New England Biolabs.

“Single-molecule visualization of a formin-capping protein ‘decision complex’ at the actin filament barbed end”

Regulation of actin filament length is a central process by which eukaryotic cells control the shape, architecture, and dynamics of their actin networks. This regulation plays a fundamental role in cell motility, morphogenesis, and a host of processes specific to particular cell types. This paper by recently graduated [Biophysics and Structural Biology] Ph.D. student Jeffrey Bombardier and collaborators resolves the long-standing mystery of how formins and capping protein work in concert and antagonistically to control actin filament length. Bombardier used the CoSMoS multi-wavelength single-molecule fluorescence microscopy technique to to discover and characterize a novel tripartite complex formed by a formin, capping protein, and the actin filament barbed end. Quantitative analysis of the kinetic mechanism showed that this complex is the essential intermediate and decision point in converting a growing formin-bound filament into a static capping protein-bound filament, and the reverse. Interestingly, the authors show that “mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex.” The results define the essential features of the molecular mechanism of filament length regulation by formin and capping protein; this mechanism predicts several new ways by which cells are likely to couple upstream regulatory inputs to filament length control.

Single-molecule visualization of a formin-capping protein ‘decision complex’ at the actin filament barbed end
Jeffrey P. Bombardier, Julian A. Eskin, Richa Jaiswal, Ivan R. Corrêa, Jr., Ming-Qun Xu, Bruce L. Goode, and Jeff Gelles
Nature Communications  6:8707 (2015)

The capping protein expression plasmid described in this article is available from Addgene.

Readers interested in this subject should also see a related article by Shekhar et al published simultaneously in the same journal.  We are grateful to the authors of that article for coordinating submission so that the two articles were published together.

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