**Abstract**

Primary cilia are sensory organelles present on the surface of most polarized cells. Primary cilia have been demonstrated to play many sensory cell roles, including mechanosensory and chemosensory functions. We demonstrated previously that primary cilia of vascular endothelial cells will bend in response to fluid shear stress, which leads to the biochemical production and release of nitric oxide. This process is impaired in endothelial cells that lack primary cilia function or structure. In this chapter, we will provide an overview of ciliogenesis and the differences between primary cilia and multicilia, as well as an overview of our published work on primary cilia and nitric oxide, and a brief perspective on their implications in health and disease.

**Keywords:** primary cilia, nitric oxide, signaling, fluid shear stress, mechanosensory transduction

## **1. Introduction**

Cilia are found in nearly every cell in the animal body, where they function as highly specialized sensory organelles. Ciliary malfunction, therefore, tends to result in severe abnormalities, which are often multisystemic. These abnormalities are known as ciliopathies, and as our understanding of cilia form and function continues to grow, so too does the list of known ciliopathies. It is now known that mutations in over 40 genes can alter cilia structure or function, and this list continues to grow; over 1000 polypeptides in the ciliary proteome have yet to be researched [1, 2].

The field of cilia research gained interest after the discovery that cilia play a role in the pathogenesis of Polycystic Kidney Disease (PKD) as fluid mechanosensors within the kidney. In addition to renal dysfunction, the cardiovascular system is also affected by PKD, which has prompted further research into the role that primary cilia play within this system. In kidney tubule epithelia, primary cilia activation leads to a calcium influx, and it has been proposed that this may also occur in vascular endothelial cells. In their study, Nauli et al. showed that vascular cilia play a similar function in sensing fluid shear stress, and there was a corresponding increase in calcium levels correlated with nitric oxide (NO) release. This is thought to contribute to blood pressure control directly. Testing this hypothesis, Nauli et al. showed that cilia mutant cell lines had little to no calcium influx, as well as a lack of NO release while under fluid shear stress [1, 3, 4].

Nitric oxide is a signaling molecule that plays many important functional roles in almost every organ system in the body. Various pathologies are associated with wayward NO production and altered bioavailability levels caused by abnormal

signaling cascades, which are often the result of abnormal cilia-regulated signaling pathways. There is a documented connection between cilia and NO in the vasculature, as well as an overlap between signaling pathways in other pathologies. It has been postulated that there is a connection between primary cilia and NO outside of the vasculature, but literature on the subject is scarce [1].

This chapter aims to explain cilia type, structure, and function, as well as ciliogenesis, nitric oxide signaling, and finally the interplay between nitric oxide and primary cilia.

### **2. Cilia type and structure**

To understand what makes primary cilia unique, it is important to understand the differences between cilia form and function. Cilia are dynamic sensory organelles present in nearly every cell in every animal, as well as most protozoa. There are two classes of cilia; motile, which possess the dynein motor complexes needed to move, and nonmotile. Motile and nonmotile cilia both contain a 25 μm diameter cytoskeletal scaffold known as the axoneme [5]. The axoneme is comprised of hundreds of proteins and houses nine peripheral microtubule doublets. These doublets are made up of A and B tubules, and they either surround a central pair of microtubules (9 + 2 pattern), or do not (9 + 0 pattern) [5]. Some motile cilia contain a 9 + 2 pattern and exist in clusters on cells called multiciliated cells (MCCs) [6]. There is also a class of motile cilia that have a 9 + 0 structure and exist as solitary monocilia on cell surfaces. The presence or absence of the central pair leads to significant functional differences in the cilia. The 9 + 2 structure commonly moves in a wave-like motion to move fluid, and an example of this are the ependymal cilia. The 9 + 2 patterned cilia also move cerebral spinal fluid, while the 9 + 0 structured most commonly moves in a rotary or corkscrew motion, as seen in flagella, which is useful for propulsion [7, 8]. There is some debate on whether sperm tail flagella should be classified as motile monocilia; regardless, they also possess a similar axonemal structure [5, 6, 9–11]. Nonmotile cilia, known as primary cilia, have a 9 + 0 structure and exist as monocilia on the surface of cells. As primary cilia can be found on vascular endothelial cells, they will be the focus of this review, but a brief overview of multicilia and their motion will also be covered.

#### **2.1 Ciliogenesis**

Cilia formation is known as ciliogenesis. Ciliogenesis is correlated with cell division and occurs at the G1/G0 phase of the cell cycle. Reabsorption or disassembly of the cilium starts after cell cycle re-entry. In the first step of ciliogenesis, the centrosome travels to the cell surface, whereupon a basal body is formed by the mother centriole, and it nucleates the ciliary axoneme at the G1/G0 phase of the cell cycle [12]. This first process is regulated by distal appendage proteins, such as centrosomal protein 164 [13]. During the second step, the cilium elongates; this process is regulated by nuclear distribution gene E homolog 1 (Nde1), up until the cilium is matured [14]. The third step is cilia resorption, followed by axonemal shortening during cell cycle reentry. This third process is controlled by the Aurora A-HDAC6, Nek2-Kif24, and Plk1-Kif2A pathways [15]. In the fourth step, the basal body is released from the cilia, which frees the centrioles that act as microtubule organizing centers (MTOC) or spindle poles for mitosis [12].

Immotile cilia formation is impacted by the coordination of the assembly and disassembly equilibrium, the IFT system, and membrane trafficking. When the axoneme nucleates from the basal body, it contains a microtubule bundle contained

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**Figure 1.**

*Primary Cilia are Sensory Hubs for Nitric Oxide Signaling*

within the ciliary membrane [16]. Enclosed within are certain signaling molecules and ion channels. Because cilia lack the machinery needed to synthesize ciliary proteins, proteins synthesized by the cell's Golgi apparatus must be transported through a ciliary 'gate' and transition zone near the cilium base [17]. The transition zone, recognizable by a change from triplet to doublet microtubules, is located at the distal end of the basal body (**Figure 1**) [18]. Basal body docking with the plasma membrane can be either permanent, in the case of unicellular organisms, or tempo-

Transition fibers, which are present in unicellular organisms, or distal and subdistal appendages, which are present in mammals, are attached to microtubules within the transition zone [19]. Transition fibers function as docking sites for intraflagellar transport (IFT) proteins [20]. IFT transports cargo in a bidirectional manner along the length of cilia, and is mediated by kinesin-2 (anterograde) and cytoplasmic dynein-2 motors (retrograde) attached to multisubunit protein complexes known as IFT particles [21, 22]. Y-linkers exist at the distal end of the transition zone and secure

the doublet microtubules to the ciliary membrane in most organisms [19].

In vertebrates, MCCs are present in a wide variety of different tissue types. In mammals, ependymal MCCs line brain ventricles and the airway epithelium. Multicilia are produced by specialized cells for highly specialized functions. MCCs are typically defined as having more than two cilia on their surface, although this occurrence is not well documented or understood. Recently, MCCs have been observed in unicellular eukaryotes and protists, as well as many metazoans, and even in certain plant sperms [23–25]. MCCs result in the production of motile axonemes, with the only notable exception being mammalian olfactory cilia. These olfactory MCCs lack dynein arms and are considered immotile despite having a 9 + 2

*Primary cilia structure. The axonemes of primary cilia are anchored from the basal body and encapsulated within the ciliary membrane. The ciliary membrane is one continuous extension of the plasma membrane. The basal body is composed of the mother and the daughter centrioles, as well as some transition fibers that anchor the basal body to the cell membrane. The ciliary membrane houses specific membrane and protein receptors, all of which facilitate proper cilia signaling (left panel). Primary cilia that are found on vascular endothelial cells are identifiable by an immunofluorescence technique with antibody against acetylated α-tubulin (green) labeling primary cilia, and pericentrin (red) labeling the centriole or basal body. The nucleus is counterstained* 

*with DAPI (blue) to label DNA (right panel). Left panel is adopted from Ref. [1].*

*DOI: http://dx.doi.org/10.5772/intechopen.89680*

rary, in the case of metazoans [5].

**2.2 Multicilia**

#### *Primary Cilia are Sensory Hubs for Nitric Oxide Signaling DOI: http://dx.doi.org/10.5772/intechopen.89680*

within the ciliary membrane [16]. Enclosed within are certain signaling molecules and ion channels. Because cilia lack the machinery needed to synthesize ciliary proteins, proteins synthesized by the cell's Golgi apparatus must be transported through a ciliary 'gate' and transition zone near the cilium base [17]. The transition zone, recognizable by a change from triplet to doublet microtubules, is located at the distal end of the basal body (**Figure 1**) [18]. Basal body docking with the plasma membrane can be either permanent, in the case of unicellular organisms, or temporary, in the case of metazoans [5].

Transition fibers, which are present in unicellular organisms, or distal and subdistal appendages, which are present in mammals, are attached to microtubules within the transition zone [19]. Transition fibers function as docking sites for intraflagellar transport (IFT) proteins [20]. IFT transports cargo in a bidirectional manner along the length of cilia, and is mediated by kinesin-2 (anterograde) and cytoplasmic dynein-2 motors (retrograde) attached to multisubunit protein complexes known as IFT particles [21, 22]. Y-linkers exist at the distal end of the transition zone and secure the doublet microtubules to the ciliary membrane in most organisms [19].
