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!

Form meets function

Professor of Biology Piali Sengupta gave the 2015 Stetten Lecture at NIGMS on Oct 21 on Form Meets Function: Structurally Diverse Cilia and Their Roles in Sensory SignalingThe “cilia squad” in the Sengupta Lab has been working for some years now to examine cilia formation in sensory neurons in the nematode C. elegans, and the relationship between the structure and nerve cell function. You can watch Piali’s lecture online.


Deep inside a worm’s nose

In a new paper in eLIFE, a team spearheaded by Brandeis postdocs David Doroquez and Cristina Berciu provide a strikingly detailed look at key structures called cilia on neurons involved in sensory perception in the nematode C. elegans. Primary cilia are the antenna-like structures onsensory neurons that gather information about the animal’s environment, such as chemicals, temperature, humidity, and touch. The genetic tools available to manipulate individual, identifiable neurons in C. elegans make worms an excellent model organism to study the assembly and function of cilia. This study requires a description of the structure of the cilia and their immediate surrounding glial support cells, and this new paper, a collaboration of the Sengupta and Nicastro labs, provides high-resolution 3D models showing how diverse and specialized these structures are.


A bouquet of sensory antennae. The 3D ultrastructure of all sensory cilia
and other neuronal projections in the head of the soil roundworm C.
elegans have been reconstructed using serial section transmission electron
microscopy. Shown are 3D isosurface-rendering models emerging from a
transmission electron microscopic cross-section of the worm.

The key techniques in this study were serial section transmission electron microscopy and electron tomography, with structures well-preserved by high-pressure freezing and freeze-substitution. With these techniques, the authors achieved the first high-resolution 3D reconstructions of 50/60 cilia from C. elegans. They describe several previously uncharacterized features — for example, there are distinct types of branching patterns – in one, the two cilia originate from independent basal bodies (as previously seen in Chlamydomonas). In the second, the cilia branch after the basal transition zone, the ciliary gatekeeper region. In the latter case, this basically means that whatever is needed for the cilia to branch has to be transported through the transition zone, suggest there might be novel mechanisms of ciliary protein trafficking. In a third pattern, the branching occurs proximally before the transition zone, and represent therefore dendritic microvilli, rather than ciliary branching. The study also showed different organizations  of microtubules in different cilia types and vesicles in regions of the cilia which have never been seen before, again pointing to new mechanisms of protein transport. They also describe new cilia-glial interactions, which might suggest that cilia and glia talk to each other.

For more about these structures (with lots of pretty pictures and movies), see:

“Similar differences”: radial spokes are different from each other, but conserved across species

Eukaryotic cilia and flagella are highly conserved organelles present on the surfaces of many animal cells and protists. These organelles are important for cell motility and/or sensing of the environment. Virtually all motile cilia and flagella share the same microtubule-based core structure, the axoneme, composed of nine doublet microtubules (DMTs) surrounding a central pair of singlet microtubules known as the central pair complex (CPC) (Fig. 1). Structurally, each DMT is built from many copies of a 96 nm-long unit that repeats along the longitudinal DMT axis (Fig. 1A). The proper assembly and function of the components of these repeat units are necessary for the generation of the characteristic wave form of ciliary and flagellar movement. Mutations in these components have been linked to various human diseases termed ciliopathies.

”]Important components of the 96 nm repeat unit include the dyneins – molecular motors that convert chemical energy into motion – and complexes that regulate dynein activity, such as radial spokes (RSs) and the Nexin-Dynein Regulatory Complex (N-DRC). The dyneins are arranged in two rows: the inner-dynein arms (IDAs) and the outer-dynein arms (ODAs) (Fig. 1B). All dynein motors are permanently anchored on one DMT and generate force by walking along the neighboring DMT, causing both DMTs to slide against one another. Connections between neighboring DMTs, e.g.the nexin links, restrict this sliding motion between DMTs and transform it into the bending motion typical of cilia and flagella. Dynein motors can only move in one direction: towards the minus end of the DMT. Consequently, dyneins on one side of the axoneme cause the axoneme to bend in one direction, whereas dyneins on the opposite side of the axoneme cause it to bend in the opposite direction (Fig. 1B).

Figure 1 (right): A) Model of the 96nm axonemal repeat containing important regulatory structures. B) An overview of the axoneme in cross section. C and D) Axoneme bending in opposing directions is achieved by alternating activation of dynein motors between opposing DMT subsets. [Heuser et al., 2009]

To generate the characteristic flagellar beating patterns, only subsets of dyneins must be active at any given time. If the dyneins were all active at once, this would put the cilia and flagella in a rigor-like state, as opposite sides of the axoneme would be trying to bend in opposing directions. Precise regulation of  dyneins present on subsets of DMTs is thus required to achieve a normal beating pattern. The current model for how cilia and flagella achieve such regulation is that the dyneins receive chemical and mechanical cues from the CPC, through the RSs, to the IDAs or through the N-DRC and the I1-dynein complex to the ODAs. The RSs are key players in this signaling pathway that regulates motility. Previously, classical electron microscopy studies have described RSs as T-shaped structures that are present either as pairs (termed RS1 and RS2; e.g. in Chlamydomonas) or triplets (termed RS1, RS2, and RS3; e.g. in mammalian cilia, sea urchin flagella, and Tetrahymena).  It was thought that RSs within pairs or triplets were all structurally identical. Recently, however, the Nicastro group and collaborator Elizabeth Smith’s lab at Dartmouth University demonstrated that RS1 and RS2 (green and blue in Fig. 2 bottom) of the RS pair in Chlamydomonas exhibit heterogeneity, i.e. their docking to the DMT and the structure of their bases are different, suggesting different roles in motility regulation (Barber et al., 2012; Dymek et al. 2011).

The Nicastro lab at Brandeis University aims at understanding the structure and function of cilia and flagella. Using state-of-the-art structural methods, such as cryo-electron tomography and sub-tomogram averaging, the Nicastro lab is visualizing the axonemes of different model organisms such as protists (e.g. the bi-flagellated alga Chlamydomonas or the ciliate Tetrahymena with thousands of cilia) and metazoa (e.g. sea urchin sperm flagella) at unprecedented detail (Nicastro et al. 2006; Heuser et al. 2009).

In a new study, Dr. Jianfeng Lin (a post-doc in the Nicastro lab) and his colleagues from the Nicastro lab have revealed a remarkably distinct RS heterogeneity that is highly conserved across species (Fig. 2) (Lin et al., 2012). At a resolution of 3.6 nm, it became obvious that RS3 (orange in Fig. 2 top) is structurally unique; bearing no resemblance to RS1 and RS2 (green and blue in Fig. 2 top). In comparison to RS1 & 2, RS3 is larger in mass, and has a bulkier and irregular shape, including the RS head, which consists of two rotationally symmetric halves in RS1 & 2, but is asymmetric in RS3 (Fig. 2 top).

Figure 2 (left): Top) Isosurface renderings of RS1 (green), RS2 (blue) and RS3 (orange) in sea urchin sperm flagella. Center) Cross section model of a sea urchin sperm flagellum displaying doublet specific features of the RSs. Bottom) Isosurface renderings of RS1 (green), RS2 (blue) and RS3S (orange) in Chlamydomonas flagella.

Perhaps the most intriguing structural difference observed was that RS3 exhibits features which are specific to only some of the nine DMTs, so called doublet-specific features (Fig. 2 center). For example, a structure termed the Radial Spoke Joist (RSJ) is only present on DMTs 3, 4, and 7-9 (magenta in Fig. 2 center). These features seem to act as a triad that connects three major regularory complexes, RS2, RS3, and the N-DRC. The doublet-specific features observed on RS3 suggest that RS3 plays a unique and currently unknown role in generating typical cilia and flagella beating patterns in sea urchin flagella and Tetrahymena cilia.

Although the Chlamydomonas axoneme contains only RS pairs, it does possess a structure that occupies the same site in each axonemal repeat that a third RS would occupy in organisms with RS triplets (orange in Fig. 2 bottom). This structure was termed the RS3-stand in (RS3S) by Barber et al. (2012). A comparison of the Chlamydomonas RS3S to RS3 in RS triplets revealed that the basal region of RS3 and the entire RS3S are virtually identical in structure (Fig. 2), suggesting that the RS3S is a shorter homolog of RS3 (Lin et al., 2012).

Although past proteomic studies identified the protein composition of the Chlamydomonas RS pair, i.e. of RS1 & 2, the new data suggest that the proteome of RS3 has yet to be determined. RS3 may play a novel role in regulating ciliary and flagellar motility and determining its protein composition in future studies should provide valuable clues to its function.

Reference: Nicastro et al. (2006) Science 313: 944-8; Heuser et al. (2009) J Cell Biol 187: 921-33; Dymek et al. (2011) Mol Biol Cell 22: 2520-31; Barber et al. (2012) Mol Biol Cell 23: 111-20; Lin et al. (2012) Cytoskeleton [Epub ahead of print].

Cryo-electron tomography and the structure of doublet microtubules

In a new paper in PNAS entitled “Cryo-electron tomography reveals conserved features of doublet microtubules“, Assistant Professor of Biology Daniela Nicastro and coworkers describe in striking new detail the structure and organization of the doublet microtubules (DMTs), the most conserved feature of eukaryotic cilia and flagella.

Cilia and flagella are thin, hair-like appendages on the surface of most animal and lower plant cells, which use these organelles to move, and to sense the environment. Defects in cilia and flagella are known to cause disease and developmental disorders, including polycystic kidney disease, respiratory disease, and neurological disorders. An essential feature of these organelles is the presence of nine outer DMTs (hollow protein tubes) that form the cylindrical core of the structure known as the axoneme. The doublet microtubule is formed by tubulin protofilaments and other structural proteins, which provide a scaffold for the attachment of dynein motors (that drive ciliary and flagellar motility) and regulatory components in a highly specific and ordered manner.

To address long-standing questions and controversies about the assembly, stability, and detailed structure of DMTs , the Nicastro lab used a high-resolution imaging technique, cryo-electron microscope tomography (cryo-ET), to probe the structure of DMTs from Chlamydomonas (single-celled algae) and sea urchin sperm flagella. Cryo-ET involves:

  1. rapid freezing of the sample to cryo-immobilize the molecules without forming ice crystals,
  2. tilting the specimen in the electron microscope to collect ~70 different views from +65° to –65°,
  3. computational alignment of the views to calculate a tomogram (a three-dimensional reconstruction of the imaged sample), and
  4. computational averaging of repeating structures in the tomogram to reduce noise and increase resolution.

Cryo-ET provided the necessary resolution to show that the B-tubules of DMTs are composed of 10 protofilaments, not 11, and that the inner and outer junctions between the A- and B-tubules are fundamentally different (see figure). The outer junction, crucial for the initial formation of the DMT, appears to be formed by interactions between the tubulin subunits of three protofilaments with unusual tubulin interfaces, but one of these protofilaments does not fit with the conventionally accepted orientation for tubulin protofilaments. This outer junction is important physiologically, as shown by mutations affecting the usual pattern of posttranslational modifications of tubulin. In contrast, the inner junction is not formed by direct interactions between tubulin protofilaments. Instead, a ladder-like structure that is clearly thinner than tubulin connects protofilaments of the A- and B-tubules.

The level of detail also allowed the Nicastro lab to show that the recently discovered microtubule inner proteins (MIPs) located within the A- and B-tubules are more complex than previously thought. MIPs 1 and 2 are both composed of alternating small and large subunits recurring every 16 and/or 48 nm along the inner A-tubule wall. MIP 3 forms small protein arches connecting the two B-tubule protofilaments closest to the inner junction, but does not form the inner junction itself. MIP 4 is associated with the inner surface of the A-tubule along the partition protofilaments, i.e., the five protofilaments of the A-tubule bounded by the two junctions with the B-tubule.

The Nicastro lab plans to build on this foundation in future work on the molecular assembly and stability of the doublet microtubule and axoneme, and hope to use it to elucidate molecular mechanisms of ciliary and flagellar motility and signal transduction in normal and disease states.

Other authors on the paper include Brandeis postdocs Xiaofeng Fu and Thomas Heuser, Brandeis undergrad Alan Tso (’10), and collaborators Mary Porter and Richard Linck from the University of Minnesota.

Microtubules and Molecular Motors Do The Wave

Most people are familiar with audiences in crowded arenas performing “the wave,” raising their hands in sync to produce a pattern that propagates around the whole stadium.  This self-organized motion appears seemingly out of nowhere.  It is not produced by any external control, but is rather emerges from thousands of individuals interacting only with their neighbors.  A similar principle of self-organization might also be relevant on length scales that are billion times smaller.  On this scale, nanometer-sized proteins interact with each other to produce dynamical structures and patterns that are essential for life—and some of these processes are reminiscent of waves in crowded stadiums.  For example, thousands of nano-sized molecular motors located within a single eukaryotic flagellum or cilium coordinate their activity to produce wave-like beating patterns.  Furthermore, dense arrays of cilia spontaneously synchronize their beating to produce metachronal waves.

Proper functioning of cilia is essential for health; for example, cilia determine the correct polarity and location of our organs during development.  Defective cilia can cause a serious condition called situs inversus, in which the positions of the heart and lungs are mirrored from the normal state.  In another example, thousands of cilia in our lungs function to clear airways of microscopic debris such as dust or smoke by organizing their beating into coordinated, wave-like patterns.  Despite the importance of ciliar function, the exact mechanisms that lead to spontaneous wave-like patterns within isolated cilia, as well as in dense ciliary fields, is not well understood.

In a paper published in the journal Science this week, an interdisciplinary team consisting of physics graduate student Timothy Sanchez and biochemistry graduate student David Welch working with biophysicist Zvonimir Dogic and biologist Daniela Nicastro present a striking finding: the first example of a simple microscopic system that self-organizes to produce cilia-like beating patterns.  Their experimental system consists of three main components: 1) microtubule filaments; 2) motor proteins called kinesin, which consume chemical fuel to move along microtubules; and (3) a bundling agent that induces assembly of filaments into bundles.  Sanchez et al. found that under a certain set of conditions, these very simple components are able to self-organize into active bundles that spontaneously beat in a periodic manner.  One large spontaneously beating bundle is featured below:

In addition to observing the beating of isolated bundles, the researchers were also able to assemble a dense field of bundles that spontaneously synchronized their beating patterns into traveling waves.  An example of this higher-level organization is shown here:

The significance of these observations is several-fold. First, due to the importance of ciliar function for health, there is great interest in elucidating the mechanism that controls the beating patterns of isolated cilia as well as dense ciliary fields.  However, the complexity of these structures presents a major challenge.  Each eukaryotic flagellum and cilium contains more than 600 different proteins.  For this reason, most previous studies of cilia and flagella have employed a top-down approach; they have attempted to elucidate the beating mechanism by deconstructing the fully functioning organelles through the systematic elimination ­­­of constituent proteins. In this study, the researchers utilize an alternative bottom-up approach and demonstrate for the first time that it is possible to construct artificial cilia-like structures from a “minimal system,” comprised of only three components.  These observations suggest that emergent properties, spontaneously arising when microscopic molecular motors interact with each other, might play a role in formation of ciliary beating patterns.

Second, self-organizing processes in general have recently become the focus of considerable interest in the physics community.  These processes range in scale from microscopic cellular functions and swarms of bacteria to macroscopic phenomena such as flocking of birds and manmade traffic jams. Theoretical models indicate that these vastly different phenomena can be described using similar theoretical formalisms.  However, controllable experiments with flocks of birds or crowds at football stadiums are virtually impossible to conduct.  The experiments described by Sanchez et al. could serve as a model system to test a broad range of theoretical predictions. Third, the reproduction of such an essential biological functionality in a simple in vitro system will be of great interest to the fields of cellular and evolutionary biology. Finally, these findings open the door for the development of one of the major goals of nanotechnology: to design motile nano-scale objects.

These encouraging results are only the first from this very new model system.  The Dogic lab is currently planning refinements to the system to study these topics in greater depth.

UPDATE: Today, this publication was additionally featured on NPR Science Friday as the video pick of the week:


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