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Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with modules to interact with the extracellular-matrix(ECM) and the intra-cellular cytoskeletal machinery. CD44 has been recently shown to compartmentalize the membrane into domains by acting as membrane pickets, facilitating the function of signaling receptors. While spatial organization and diffusion studies of membrane proteins are usually conducted separately, here we combine observations of organization and diffusion by using high spatio-temporal resolution imaging on living cells to reveal a hierarchical organization of CD44. CD44 is present in a meso-scale meshwork pattern where it exhibits enhanced confinement and is enriched in nano-clusters of CD44 along its boundaries. This nanoclustering is orchestrated by the underlying cortical actin dynamics. Interaction with actin is mediated by specific segments of the intracellular-domain(ICD). This influences the organization of the protein at the nano-scale, generating a selective requirement for formin over Arp2/3-based actin-nucleation machinery. The extracellular-domain(ECD) and its interaction with elements of ECM do not influence the meso-scale organization, but may serve to reposition the meshwork with respect to the ECM. Taken together, our results capture the hierarchical nature of CD44 organization at the cell surface, with active cytoskeleton-templated nano-clusters localized to a meso-scale meshwork pattern.

Hedgehog (Hh)-containing exovesicles are mediators of long-range signaling in Drosophila wing imaginal discs. ESCRT-dependent multivesicular body (MVB) biogenesis is essential for generating Hh exovesicles. Oligomerization of Hh at the cell surface is necessary for its endocytic delivery to MVBs and therefore is required for its release as exovesicles., Hedgehog (Hh) is a secreted morphogen involved in both short- and long-range signaling necessary for tissue patterning during development. It is unclear how this dually lipidated protein is transported over a long range in the aqueous milieu of interstitial spaces. We previously showed that the long-range signaling of Hh requires its oligomerization. Here we show that Hh is secreted in the form of exovesicles. These are derived by the endocytic delivery of cell surface Hh to multivesicular bodies (MVBs) via an endosomal sorting complex required for transport (ECSRT)–dependent process. Perturbations of ESCRT proteins have a selective effect on long-range Hh signaling in Drosophila wing imaginal discs. Of importance, oligomerization-defective Hh is inefficiently incorporated into exovesicles due to its poor endocytic delivery to MVBs. These results provide evidence that nanoscale organization of Hh regulates the secretion of Hh on ESCRT-derived exovesicles, which in turn act as a vehicle for long-range signaling.

Membrane proteins that couple to cortical actin show temperature-independent diffusion. The loss of this coupling and perturbation of cortical actomyosin dynamics render the diffusion temperature dependent. The findings suggest that active fluctuations arising from dynamic actin filaments at the cortex can drive diffusion on the cell membrane., Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24–37°C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an “active actin-membrane composite” cell surface.

A diverse set of cell surface eukaryotic proteins including receptors, enzymes, and adhesion molecules have a glycosylphosphoinositol-lipid (GPI) modification at the carboxy-terminal end that serves as their sole means of membrane anchoring. These GPI-anchored proteins are poorly solubilized in nonionic detergent such as Triton X-100. In addition these detergent-insoluble complexes from plasma membranes are significantly enriched in several cytoplasmic proteins including nonreceptor-type tyrosine kinases and caveolin/VIP-21, a component of the striated coat of caveolae. These observations have suggested that the detergent-insoluble complexes represent purified caveolar membrane preparations. However, we have recently shown by immunofluorescence and electron microscopy that GPI-anchored proteins are diffusely distributed at the cell surface but may be enriched in caveolae only after cross-linking. Although caveolae occupy only a small fraction of the cell surface (< 4%), almost all of the GPI-anchored protein at the cell surface becomes incorporated into detergent-insoluble low-density complexes. In this paper we show that upon detergent treatment the GPI-anchored proteins are redistributed into a significantly more clustered distribution in the remaining membranous structures. These results show that GPI-anchored proteins are intrinsically detergent-insoluble in the milieu of the plasma membrane, and their co-purification with caveolin is not reflective of their native distribution. These results also indicate that the association of caveolae, GPI-anchored proteins, and signalling proteins must be critically re-examined.

I must confess, I came to cell biology rather serendipitously; perhaps my journey may carry some ideas for young scientists who want to prepare themselves for studying cell biology today. Although I was interested in biology as a high school student, I found myself as an undergraduate at the Indian Institute of Technology, studying to become an engineer. Fortunately for me, the chemistry department was offering a few courses in biological chemistry, and I quickly decided to go through with the initial grounding (we used to call it grinding) in basic physics, chemistry, mathematics and engineering sciences, before joining the chemistry program with a specialization in bio-organic chemistry. For my Ph.D., I chose a biological subject, the mechanism of GPI-anchoring of the Variant Surface Glycoprotein, the protective coat of the protozoan pathogen that causes African sleeping sickness. The idea then was to identify a pathogen-specific pathway that can be targeted to cure this devastating disease—a goal that seems hopelessly naive now that we know that the GPI-anchor is made in almost all eukaryotes via a highly conserved pathway and that it has important cellular functions. Satyajit Mayor Having been exposed to the world of exotic biomolecules, my training in chemistry and physics led me to explore how these molecules manufactured inside a cell would behave in a cellular context and to examine more general questions of evolutionary commonality of cellular processes and behavior. I have since focused on GPI-anchored proteins as well as many other lipids and lipid-anchored proteins, in their “natural habitat,” the plasma membrane of the mammalian cell, a more tractable system than the parasite cell membrane. I realize now that this serendipitous path has been quite instrumental in shaping many of the questions I am asking today. Being open-minded about interesting observations other than those one may have been expecting, coupled with a firm grounding in the physical and natural sciences and a healthy respect for what could happen in the very special cellular environment, form a good base (I believe) for studying cell biology, a field still full of tantalizing mysteries. One of the greatest mysteries is that although constituents in a cell are never static and every structure, configuration, and organelle is actively turning over, a cell retains a consistent form (phenotype) and makes reproducible physiological responses in a given niche. This occurs over different time (milliseconds to hours if not longer) and spatial (nanometer to hundreds of microns) scales, for example, in the construction of signaling complexes at the cell surface, or in the precise three-dimensional arrangement of chromosomes inside a eukaryotic nucleus, in response to a given niche. Elucidating how cells reproducibly put molecules together may depend on understanding nonequilibrium processes that are maintained by continuous energy consumption and mechanisms that violate detailed balance. Take the cell membrane, a cellular material that I happen to be fascinated with. Insights about this complex milieu will follow from our understanding of how the cell is able to regulate the local configuration of molecules in a two-dimensional fluid matrix, in the face of thermal dissipation and the thermodynamic propensities of the molecules embedded in this physical medium. To understand this complex system, we will need to build robust theoretical frameworks crossing traditional interdisciplinary boundaries, notably to encompass information theory and evolutionary biology, incorporating new ideas of active systems that are being explored in soft-matter physics. Like chemistry, before insights of the atomic theory explained why elements had their particular physical and chemical properties, cell biology is still a largely phenomenological discipline, which is why it is essential to remain open to serendipitous observations and to seek insights, wherever they can be profitably found. I was fortunate to return to work to India when it was beginning to undergo a major positive transformation in government policy toward science funding (Mayor, 2005 ). Cell biology is as yet nascent in India, but the potential is huge; funding continues to be generous, and there are no restrictions on the scope of exciting basic scientific questions that can be addressed. Exactly how the cell biology community evolves will depend on maintaining the current collaborative spirit and encouraging interdisciplinary academic research and exploration of science for its own sake, as well as attracting talent to take up the challenge of charting new territory in India. This will in turn depend on how young scientists are trained—perhaps the place of serendipity in the history of science and the importance of being receptive to diverse inputs deserves a mention.