(4fm) Biophysical Principles of Cell Organization: Engineering Condensates for Biomedical Applications

Background

The cell relies on various mechanisms to organize its contents, such as membranes and cytoskeletal filaments. Phase separation is now emerging as a powerful paradigm in cell organization: cellular components de-mix from their surroundings and self-assemble into cohesive, yet dynamic, droplets. Collectively, these droplets are called biomolecular condensates, representing distinct phases in the cell. Little is known regarding the biomolecular and biophysical features that drive cellular components to condense into higher-order structures and how their resulting material properties contribute to their function.

My research over the past decade has centered on fundamental principles of cell organization across length and time scales, especially regarding biomolecular condensates. As a graduate student, I studied how nuclear multiphase condensates are stabilized in large frog eggs. More recently as a postdoc, I have focused on the organization and function of mitochondrial transcriptional condensates, including in the context of premature human aging. My future research will be framed around how biomolecular and biophysical interactions between proteins and nucleic acids encode spatiotemporal information to give rise to condensed phases in cells, and how they can be engineered to control cell organization and function, ultimately aiming to reverse disease.

Condensate organization in large cells

Most cells are typically ~1-100 microns in size, unable to be seen by eye. Biology always has a few exceptions, such as frog eggs that reach remarkable sizes of 1 mm – orders of magnitude larger in volume than a typical cell. Using microrheology to probe the material properties of these large frog nuclei, I discovered the presence of a viscoelastic actin network, suggesting that the frog nucleus was not simply a bag-of-molecules, but was comprised of a mechanical scaffold [1,2].

Disruption of actin significantly altered the organization of the nucleus. The wildtype frog nucleus contains hundreds of liquid-like condensates called nucleoli. Upon actin disruption, nucleoli rapidly sedimented to the bottom of the nucleus and underwent large-scale fusion events, forming massive droplets. These results show that nuclear actin stabilized the contents against gravity, revealing that gravity becomes a dominant force as cells approach large sizes, whereas thermal motion is sufficient to organize and distribute contents at smaller length scales. The frog nucleus represents an emulsion of biomolecular condensates, kinetically stabilized by a soft, viscoelastic actin network [1,2].

As actin disruption led to fusion of nucleoli into large droplets, their internal organization could be spatially resolved. Nucleoli are well-known to have a tri-partite organization: an innermost fibrillar center (FC), surrounded by an intermediary dense fibrillar component (DFC) and an outer granular component (GC). While the nucleolus as a whole behaved as a liquid, reconciling how the nucleolus maintains its tri-partite organization was challenging. Taking advantage of the actin-disruption conditions, I captured how the nucleolus rearranged during these large-scale fusion events: each compartment underwent homotypic fusion events, taking on a drop-within-drop morphology [3]. Such behavior suggests that each compartment behaves as a separate, partially immiscible, liquid phase. To test this idea, the individual components formed similar layered structures both in vitro and in silico, driven by differences in surface tension between each liquid phase. These data prove that the nucleolus represents a multiphase, compound droplet. This new biophysical framework answers a long-standing question pertaining to the mechanism of nucleolar organization as first observed in the 1800s and has fundamental implications for the heterogenous organization of many other cellular condensates.

Mitochondrial transcriptional condensates

Phase separation is not limited to the nucleus. I showed that phase separation also organizes the contents within the mitochondria – organelles that generate energy used for the majority of cellular processes. Mitochondria are surrounded by a double membrane, but contain an inner matrix comprised of many granules, including hundreds of copies of the mitochondrial (mt-) genome. The mt-genome (~16 kb, circular) is packaged into membrane-less nucleoprotein structures called mt-nucleoids. Normally, these structures are 100 nm in size, but I found they can fuse together to form larger structures – reminiscent of the behavior of liquid droplets – especially under conditions of stress and in a premature aging disease [4].

I tested the hypothesis that the mt-genome assembles via phase separation by taking an in vitro approach. I demonstrated that the main mt-nucleoid packaging protein TFAM phase separates on its own to form viscoelastic droplets, and that mtDNA readily partitions into these droplets, influencing the material properties. Testing a variety of mutants, I found that TFAM has surfactant-like behavior, where one half preferentially interacts with mtDNA while the other half is more miscible with mt-proteins, thereby solubilizing the various components in the mt-nucleoid [4].

These mt-droplets can also be made functionally active by triggering transcription. The highly viscoelastic nature of these droplets counters the benefits of increased concentrations, leading to dampened transcriptional rates. However, transcription also affects droplet morphology leading to a transition into vesicle-like morphologies associated with increased RNA production. Collectively, these results suggest that phase separation is an evolutionarily conserved principle of genome organization and function [5].

Future research goals

Phase separation is a ubiquitous organizational principle in cells as numerous membrane-less structures, including genomes, exhibit signatures of phase behavior. Using a quantitative, engineering approach, I will study how phase separation influences cell organization across scales (molecular, organellar, and cellular) and how anomalies contribute to disease processes, including premature aging and neurodegeneration. The overarching goals are to understand the biophysical and biochemical rules that influence condensate assembly, organization, and function, and to use this knowledge to design and engineer condensates to prevent and reverse disease phenotypes.

To do so, I will use advanced approaches including genetic (including optogenetics), protein, and cellular engineering; super-resolution microscopy techniques; next-generation sequencing; and physical modeling. I will combine bottom-up in vitro approaches with in vivo behavior of various native and engineered cell types. Motivated by changes to condensate organization of mitochondrial and nuclear condensates in fibroblasts from premature aging and neurodegeneration-afflicted patients, I will develop hypothesis-driven, multidisciplinary approaches to identify biophysical mechanisms that disrupt the maintenance of liquid-like condensates, leading to aberrant organization or pathological aggregation. In conjunction, I will use the frog oocyte to understand how these “immortal” and large cells adapt to maintain their unique organization, and how these biophysical processes differ from those of their somatic counterparts, focusing on their mt-genomes and meiotic chromosomes. These approaches will shed light on the biophysical nature of condensates, opening up many directions for future work and illuminating how therapeutics and pharmaceuticals can be targeted to condensates to affect their organization and function.

Teaching Interests

Philosophy

My research experiences have straddled engineering disciplines and the biomedical sciences, reflecting different schools of thought and approaches to science, cementing the adage of “study nature, not books.” This ideology is important for new trainees, reminding them not to place great importance on rote memorization alone, but to rather learn by doing, especially when there is still so much to uncover in highly active cells! This approach deeply stirs creative thinking and curiosity, while emphasizing that nature is not bound or discretized to specific subjects such as “biology” or “physics,” but is a continuum and culmination of all these fascinating stirrings. As such, I also want to underscore clear communication for trainees, which is increasingly important as research is becoming more inter-disciplinary, blurring the boundaries between what were seemingly disparate disciplines.

I will bridge these approaches from research to learning in the classroom. I will strive to make lectures seem as a discovery process – where the ideas and topics covered are driven by questions and are packaged as tidy stories or vignettes. I hope to actively engage students with short breaks of small discussions interspersed throughout the lecture. I want to continuously challenge students to understand how the material fits into larger contexts of science and world-scale issues and not to simply get lost in a series of isolated facts.

Interests

My teaching interests revolve around fundamental principles underlying the behavior of cells. For core engineering curriculum, these would include reaction kinetics, transport processes, and thermodynamics. For technical electives, I will synthesize advanced ideas and technologies from biochemical-, cellular-, genetic-, and materials engineering.

Teaching & Mentoring Experience

I have been an undergraduate teaching fellow at UMD and a graduate assistant in instruction at Princeton for several engineering courses. I also have experience with lecture design, where I was a guest lecturer for Prof. Brangwynne’s Soft Living Matter course at Princeton. Mentoring was an immanent activity during my graduate experience as I mentored six undergraduates and one high school student for their senior theses, summer research experience and/or work study positions. These students all came from diverse personal and academic backgrounds, and I aspire to mentor the next generation of scientists, especially those from traditionally underrepresented groups.

References

1. Feric, M. and Brangwynne, C.P. (2013) Nature Cell Biology 15, 1253-1259
2. Feric, M. et al. (2015) Scientific Reports 5, 1-12
3. Feric, M. et al. (2016) Cell 165, 1686-1697
4. Feric, M. et al. (2021) The EMBO Journal 40, e107165
5. Feric, M. and Misteli, T. (2021) Trends Cell Biol. 10.1016/j.tcb.2021.03.001